Planta (1991)185:432-439
P l ~ n m 9 Springer-Verlag1991
Glucan-phosphorylase forms in cotyledons of Pisum sativum L.: Localization, developmental change, in-vitro translation, and processing Joachim van Bcrkel**, Jutta Conrads-Strauch***, and Martin Steup*
Institut ffir Botanik, WestfZ,ilische Wilhelms-Universit/itMfinster, Schlossgarten 3, W-4400 Mfinster, Federal Republic of Germany Received 24 April; accepted 10 July 1991
Abstract. The occurrence, location, and biosynthesis of glucan-phosphorylase (EC 2.4.1.1) isoenzymes were studied in cotyledons of developing or germinating seeds of P i s u m s a t i v u r n L. Type-I and type-II isoenzymes were detected, and were also localized by indirect immunofluorescence using polyclonal anti-type-I or anti-type-II phosphorylase antibodies. Type-I isoenzyme was found in the cytosol of parenchyma cells whereas the type-II enzyme form is a plastid protein which resides either in amyloplasts (in developing seeds) or in proplastids (in germinating seeds). During seed development, type-II phosphorylase was the predominant isoenzyme and the type-I isoenzyme represented a very minor compound. During germination, the latter increased whilst type-II phosphorylase remained at a constant level. In in-vitro translation experiments, type-I isoenzyme was observed as a final-size product with an apparent molecular weight of approx. 90 kDa. In contrast, type-II phosphorylase was translated as a high-molecular-weight precursor (116 kDa) which, when incubated with a stromal fraction of isolated intact pea chloroplasts, was processed to the size of the mature protein (105 kDa). Key words: Glucan phosphorylase cessing - P i s u m Starch metabolism
Isoenzyme pro-
Introduction
a-l,4-Glucan phosphorylase (EC 2.4.1.1) catalyses the reaction a-D-glucose 1-phosphate+ Gn ~ ~orthophosphate + G +1 * To whom correspondence should be addressed ** Present address: Max-Planck-lnstitutf/ir Z/ichtungsforschung, Carl-von-Linn6-Weg 10, W-5000 K61n 30, Federal Republic of Germany *** Present address." John Innes Institute, Colney Lane, Norwich NR4 7UH, UK Abbreviations." IgG = immunoglobulin G; kDa = kilodalton; poly(A)+RNA = polyadenylated R N A ; SDS-PAGE= sodium dodecyl sulfate-polyacrylamidegel electrophoresis
where G and G,+ 1 represent an a-glucan composed of n and (n + 1) a-l,4-1inked glucosyl moieties, respectively. The enzyme is widely distributed in microorganisms, animals, and plants. Multiplicity is a common feature of glucan phosphorylase. In mammals, three phosphorylase isoenzymes occur (in muscle, liver, and brain) which are preferentially expressed in the respective organ and, thereby, give rise to an organ-specific pattern of phosphorylase isoenzymes (Newgard et al. 1989). Higher plants contain, within the same cell, phosphorylase isoenzymes which differ in subunit size and kinetic properties, especially in glucan specificity (Nakano and Fukui 1986; Steup 1988). One higher-plant phosphorylase (type I) has apparent monomer size of approx. 90 kDa and exhibits a relatively low affinity towards maltodextrins. Its affinity towards branched polyglucans, such as soluble starch or glycogen, is high (Steup and Schfichtele 1981; Shimomura et al. 1982). In leaves, this enzyme form resides in the cytosol of mesophyll cells (Conrads et al. 1986; Schfichtele and Steup 1986). The other phosphorylase form (type II) is restricted to the stromal space of chloroplasts (Sch/ichtele and Steup 1986), has a monomer size of more than 100 kDa, and possesses a high affinity towards maltodextrins but an extremely low affinity towards highly branched glucans, such as glycogen. In leaflets of P i s u m s a t i v u m L., two chloroplastic (type II) phosphorylase forms have been observed which are similar in size and kinetic properties (Steup and Latzko 1979; Steup et al. 1986). For the plastidic potato-tuber phosphorylase form (type-II isoenzyme), both the larger monomer size and the glucan specificity have been attributed to the existence of a unique insertion (78 amino-acid residues) which is positioned near the N-terminal/C-terminal domain junction and, possibly, impedes the binding of highly branched polysaccharides to the active site (Nakano and Fukui 1986). The metabolic implications of the dual intracellular location of higher-plant phosphorylases remain to be clarified. Two phosphorylase isoenzymes have also been observed in prokaryotes (Palm et al. 1985; Yu et al. 1988). However, both enzyme forms differ significantly from type I/type II phosphorylases of higher plants.
J. van Berkel et al.: Glucan phosphorylases in pea cotyledons I n this c o m m u n i c a t i o n , the o c c u r r e n c e a n d b i o s y n thesis o f t y p e - I a n d t y p e - I I p h o s p h o r y l a s e s h a v e been s t u d i e d in p e a c o t y l e d o n s f r o m either g r o w i n g seeds o r g e r m i n a t i n g seedlings. D e p e n d i n g u p o n the d e v e l o p m e n t a l stage, c o t y l e d o n s fulfil e n t i r e l y different m e t a bolic f u n c t i o n s : D u r i n g seed d e v e l o p m e n t , c o t y l e d o n s f u n c t i o n as a m a j o r c a r b o n sink a n d a c c u m u l a t e large q u a n t i t i e s o f starch. O n c e g e r m i n a t i o n is initiated, c o t y l e d o n s m o b i l i z e reserve s t a r c h a n d , t h e r e b y , act as the m a j o r c a r b o n source o f the entire seedling. U s i n g c o t y l e d o n s o f the v a r i o u s stages, t y p e - I a n d t y p e - I I p h o s p h o r y l a s e s were identified, quantified, a n d l o c a l i z e d in situ. T h e b i o s y n t h e s e s o f t y p e - I a n d t y p e - I I p h o s p h o r y lases were s t u d i e d b y i n - v i t r o t r a n s l a t i o n .
Material and methods Plant material Pea (Pisum sativum L. cv 'Kleine RheinlS.nderin'; Nebelung, Mfinster, FRG) seeds were surface-sterilized with sodium hypochlorite (3 % [w/v]; 60 min), washed with H20 for 4 h and were then germinated in Vermiculite under controlled conditions (Steup and Latzko 1979). Pea leaflets were harvested from 12- to 14-d-old plants grown under the same conditions. Cotyledons from developing seeds were taken from field-grown plants. Phosphorylase patterns. Extracts of pea leaflets, cotyledons or pod walls were prepared as described elsewhere (Steup 1990). Phosphorylase isoenzymes were resolved by a non-denaturing continuous electrophoresis system and were stained for glucose-l-phosphatedependent glucan-synthesizing activity as previously described (Steup 1990).
Purification of phosphorylase &oenzymes. Type-I phosphorylase was purified from pea leaflets or cotyledons (6 d after imbibition) essentially as described elsewhere (Conrads et al. 1986). Type-II isoenzyme was purified from dry seeds. The entire isolation procedure consisted of an ammonium-sulfate precipitation, ionexchange chromatography, and affinity chromatography. Dry seeds t00 g were ground to a fine powder. After the addition of 400 ml 0.1 M imidazol buffer (pH 7.0) the resulting homogenate was stirred for 15 min and centrifuged (10 min at 25 000 9). The supernatant was subjected to an ammonium-sulfate precipitation (40 to 70% saturation). The protein fraction was dissolved in 20 mM citrate buffer (pH 6.5) and dialysed against the same buffer. The sample was then applied to a diethyl aminoethyl (DEAE)-Sephacel column (5.3 cm 2 x 17.5 cm; flow 11 cm - h-1; Pharmacia, Freiburg, FRG). The column was washed with 100 ml citrate buffer and then with a linear citrate gradient ( 20 to 60 mM, pH 6.5 ; 100 ml each) to elute type-I isoenzyme (which was discarded). Type-II enzyme was eluted with a linear NaC1 gradient (0 to 0.5 M, dissolved in 60 mM citrate, pH 6.5). The enzyme preparation was then applied to an affinity gel (Sepharose-starch, deactivated with butylamine; for synthesis see Steup 1990) and eluted using a linear dextrin gradient (for details see Steup 1990). The purified type-II isoenzyme was separated from the eluents by gel filtration on Sephadex G-100 (Pharmacia; see Sch/ichtele and Steup 1986). Throughout the purification, temperature was kept at 4~ C. All media contained 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 15 gM thymol. Polyclonal antibodies. Polyclonal antibodies directed against type-I or type-II phosphorylase were raised in rabbits (Conrads et al. 1986). The anti-type-I phosphorylase immunoglobulin G (IgG) preparation did not contain idiotypes which cross-react noticeably with type-II isoenzyme (Conrads et al. 1986). Anti-type-II-phosphorylase antibodies contained a low proportion of idiotypes crossreacting with the heterologous antigen (i.e. type-I isoenzyme). These
433 idiotypes were removed from the IgG preparation as described elsewhere (Steup 1990). Immunoelectrophoresis was performed using 1% (w/v) agarose gels containing 25 mM Tricine, adjusted to pH 8.6 with Tris. Following electrophoresis (25 h at 120 V constant voltage), gels were carefully washed in 1.7% (w/v) NaCI. Precipitates were transferred to nitrocellulose and immunostained according to Hanson (1988). Indirect immunofluorescence was performed as previously described (Sch/ichtele and Steup 1986) except that the plant material was fixed in 6% (w/v) formaldehyde (overnight at room temperature) and the vacuum infiltration was omitted.
In-vitro translation and processin#. RNA was isolated from cotyledons of developing or germinating seeds according to Cashmore (1982). Polyadenylated RNA (poly(A)+RNA) was obtained by chromatography on poly U-Sepharose (Pharmacia). For size fractionation a sucrose-density-gradient centrifugation was applied (Hershey and Quail 1986). In-vitro translation was performed for 80 min at 30~ C using a rabbit reticulocyte lysate (N 90 ; Amersham, Braunschweig, FRG). Translation mixtures contained 0.1 to 0.2 lag mRNA - gl-1. For immunoprecipitation, three procedures (designated as A, B, and C) were applied. Procedure A was a modification of the technique described by Chua and Schmidt (1978) and Westhoff and Zetsche (1981). Procedure B was according to Anderson and Blobel (1983) and procedure C was according to yon Figura et al. (1985) and Conary et al. (1986). Precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE; Laemmli 1970) as modified by Rittenhouse and Marcus (1984). Proteins were transferred to nitrocellulose (Towbin et al. 1979) and were detected by autoradiography or staining with Ponceau S. For in-vitro processing a stromal fraction of isolated intact pea chloroplasts was prepared according to Robinson and Ellis (1984) and Gupta and Beevers (1987). Isolated chloroplasts were purified by centrifugation through 35% (v/v) Percoll (Pharmacia) and were broken in 20 mM Tris-HCl, pH 7.6. Proteins precipitated with ammonium sulfate (40 to 70% saturation) were dissolved in the same buffer and were desalted by filtration through Sephadex G 50 using spun columns. The protein fraction was used for processing either immediately or was stored frozen at - 70 ~ C. Processing was performed for 1 h at 27 ~ C according to Robinson and Ellis (1984) and Gupta and Beevers (1987). The processing mixture contained 30 lal translation mixture, 60 gl buffer (100 mM Hepes-KOH, pH 8.5; 220 mM KCI; 6 mM MgC12) and stromal preparation as indicated. In control mixtures, the latter was replaced by an equal volume of 20 mM Tris-HC1, pH 7.6. Phosphorylase isoenzymes were immunoprecipitated by procedure C.
Results Glucan~phosphorylase
isoenzymes
in pea
cotyledons.
Type-I and type-II phosphorylases from higher plants can be d i s t i n g u i s h e d b y affinity e l e c t r o p h o r e s i s u s i n g a g l y c o g e n - c o n t a i n i n g s e p a r a t i o n gel ( S t e u p 1990). F o r c o m p a r i s o n , e l e c t r o p h o r e s i s is p e r f o r m e d ( u n d e r o t h e r wise i d e n t i c a l c o n d i t i o n s ) in a g l u c a n - f r e e gel. O v e r a wide r a n g e o f c o n c e n t r a t i o n s , i m m o b i l i z e d g l y c o g e n d o e s n o t n o t i c e a b l y affect the m i g r a t i o n velocity o f t y p e - I I i s o e n z y m e s b u t it s t r o n g l y r e t a r d s t y p e - I enzymes: By using these criteria, t w o o f the three p h o s p h o r y l a s e s w h i c h a r e p r e s e n t in p e a leaflet e x t r a c t s were identified as t y p e - I I i s o e n z y m e s (Fig. 1A, lanes a, d). B o t h t y p e - I I e n z y m e f o r m s were o b s e r v e d in p r e p a r a t i o n s o f i s o l a t e d i n t a c t c h l o r o p l a s t s (Fig. 1A, lanes b, c). H o w e v e r , c h l o r o plasts l a c k e d the t h i r d p h o s p h o r y l a s e f o r m w h i c h is s t r o n g l y r e t a r d e d in a g l y c o g e n - c o n t a i n i n g s e p a r a t i o n gel
434
J. van Berkel et al.: Glucan phosphorylases in pea cotyledons
Fig. 2. Rocket immunoelectrophoresis of pea phosphorylase isoenzymes. Equal amounts of cotyledons of seeds 1, 3, or 6 d after soaking were homogenized in 0.1 M Tricine-Tris buffer (pH 8.6). All extracts were adjusted to the same final volume. The agarose gels contained either anti-type-l-phosphorylase IgG (left; anti-CP) or anti-type-II-phosphorylase IgG (right; anti-PP). Following electrophoresis, precipitates were transferred to nitrocellulose and were immunostained
Table 1. Type-I and type-II phosphorylase izoenzymes in extracts of cotyledons of germinating (A) or developing (B) pea seeds. Cotyledons were homogenized in 0.1 M Tricine-Tris buffer (pH 8.6). Phosphorylase isoenzymes were quantified by rocket immunoelectrophoresis. A Cotyledons were harvested 1, 3, or 6 days after soaking. B Three developmental stages were distinguished (a, b, and c) on a fresh-weight basis Fw (mg 9cotyledon - 1) Fig. 1.A Pea phosphorylase patterns. Phosphorylase isoenzymes of leaflets (lanes a, d), isolated chloroplasts (lanes b, e), cotyledons of germinating seeds (lane e: 1 d after soaking; lane f : 6 d after soaking), and pod walls (lane g) were resolved by non-denaturing continuous PAGE and subsequent activity staining. The separation gel (8.5% [w/v] total monomer concentration) was glycogen-free ( -- G) or contained 0.1% [w/v] glycogen ( + G). Approximately 70 lag protein was applied to each lane. Migration direction was from top to bottom. The arrowheads mark type-I isoenzyme which is retarded by immobilized glycogen. B Transverse gradient electrophoresis (5 to 14% (w/v) T) of extracts of pea leaflets (L) and cotyledons (C) of germinating seeds (3 d after soaking) or a 1:1 mixture of both extracts (L + C). Approximately 40 lag protein was applied to each slot. Conditions as in A
(Fig. 1A). This n o n - c h l o r o p l a s t p h o s p h o r y l a s e represents a t y p e - I i s o e n z y m e . N o n - l e a f tissues o f P i s u m satir u m c o n t a i n b o t h t y p e - I a n d t y p e - I I p h o s p h o r y l a s e s alt h o u g h their e n z y m e p a t t e r n o f t e n is less c o m p l e x t h a n t h a t o f leaflets. I n e x t r a c t s o f p e a c o t y l e d o n s two p h o s p h o r y l a s e f o r m s were r e s o l v e d b y affinity electrop h o r e s i s . O n e o f t h e m was r e t a r d e d b y i m m o b i l i z e d glyc o g e n (Fig. 1A, lanes e, f). T h e f a s t e r - m o v i n g t y p e - I I p h o s p h o r y l a s e (which is p r e s e n t in leaflets, cf. Fig. 1A, lanes a, d) was n o t d e t e c t a b l e in c o t y l e d o n s . I n p o d walls,
Isoenzyme (lag 9(mg buffer-soluble protein) - 1) Type I
Type I/ Type II
Type II
A day 1 day 3 day 6
200 220 210
0.77 2.57 7.8
2.8 2.7 3.8
0.25 0.92 2.05
60 290 700
1.5 0.7 0.3
32.8 54.3 52.1
0.04 0.01 0.006
B
stage a stage b stage c
the l a t t e r e n z y m e f o r m was o b s e r v e d as a c o m p o u n d o f m i n o r activity (Fig. 1A, lane g) w h e r e a s the m a i n b a n d s were the s a m e as t h o s e o f the c o t y l e d o n s . In a n o t h e r e x p e r i m e n t , the p h o s p h o r y l a s e f o r m s o f p e a leaflets a n d o f c o t y l e d o n s were resolved b y t r a n s v e r sal g r a d i e n t e l e c t r o p h o r e s i s using a g l y c o g e n - f r e e s e p a r a tion gel (Fig. 1B). A t a n y gel c o n c e n t r a t i o n , the t y p e - I p h o s p h o r y l a s e s o f b o t h extracts coincided. Likewise, the t y p e - I I i s o e n z y m e o f c o t y l e d o n s was e l e c t r o p h o r e t i c a l l y indiscernible f r o m the s l o w e r - m o v i n g t y p e - I I e n z y m e f o r m o f leaflets. Thus, it is highly likely t h a t the two p h o s p h o r y l a s e p r o t e i n s are identical in b o t h o r g a n s .
J. van Berkel et al.: Glucan phosphorylases in pea cotyledons
435
Fig. 3A-I. In-situ localization of type-I and type-II phosphorylase isoenzymes i cotyledons of germinating (6 d after soaking; A-F) or developing (stage b, cf. Table l ; G-I) pea seeds. Cryosections were incubated with either anti-type-Iphosphorylase IgG (D, F, I) or with anti-type-II-phosphorylase IgG (B, C, E, H) or with preimmune IgG (A, G). Magnificatons: A-D • 170; E-I, • 430. c Paraveinal tissue; A, B, D-I parenchyma cells of the cotyledons
In pea cotyledons, the ratio of type-I to type-II isoenzyme depends upon the developmental stage of the organ. The two phosphorylase forms were quantified by rocket immunoelectrophoresis followed by an electrotransfer to nitrocellulose. This modification of the conventional immunoelectrophoresis technique allows for a quantification of low-abundance proteins (Hansen 1988). Immunoelectrophoresis was performed using polyclonal anti-type-I or anti-type-II IgG. Each of the two phosphorylase isoenzymes could be monitored without interference of the heterologous antigen. This was confirmed
by control experiments in which varying amounts o f purified type-I or type-II phosphorylases were added to a crude extract o f cotyledons. The addition o f the heterologous enzyme form did not affect the immunochemical measurement o f the homologous phosphorylase protein (data not shown). In cotyledons of germinating peas, type-II phosphorylase remained at a constant level whereas the a m o u n t of type-I enzyme increased severalfold (Fig. 2). In contrast, cotyledons of developing seeds contained far more typeII phosphory!ase than type-I isoenzyme (Table 1).
436 Immunochemical localization o f phosphorylase isoenzymes. The intracellular location of type-I and type-II
phosphorylases was determined by indirect immunofluorescence. Cryosection of formaldehyde-fixed cotyledons of germinating or developing pea seeds were incubated with either anti-type-I- or anti-type-II-phosphorylase IgG (or with preimmune IgG) and, subsequently, with the fluorescein-isothiocyanate (FITC) conjugate at an appropriate dilution. Both phosphorylase isoenzymes were detected throughout the parenchyma of the cotyledons but they differed in their intracellular distribution. In cotyledons of germinating seeds, type-II phosphorylase was restricted to small proplastid-like organelles
J. van Berkel et al.: Glucan phosphorylasesin pea cotyledons which exhibited a strong fluorescence (Fig. 3b, e). These organelles were observed in the entire parenchyma tissue but they were more abundant in paraveinal cells (Fig. 3c). No fluorescence was detectable in the vicinity of the reserve starch granules. Type-I phosphorylase was observed in the entire cytosolic space of the parenchyma cells (Fig. 3d, f). In cotyledons of developing seeds, type-I phosphorylase was detected as a weak fluorescence situated in the cytosol of parenchyma cells. Neither intensity nor distribution of the immunolabel changed appreciably during seed development. Compared to type-I phosphorylase, immunolabeling of type-II isoenzyme resulted in a higher intensity of the fluorescence signal. Type-II immunolabel was restricted to the stromal space of amyloplasts. Depending upon the size of the starch granule(s) relative to that of the amyloplast, the stromal space of the organelles varied. During early stages of development, particulate starch occupied a minor proportion of the amyloplast volume and immunolabel was almost equally distributed in the entire organelle (data not shown). As starch accumulation proceeded, type-II phosphorylase isoenzyme was detected as a narrow strongly fluorescent area between the amyloplast envelope and the starch granule(s) which then occupied most of the amyloplast volume (Fig. 3h). In-vitro translation and processing o f phosphorylase isoenzymes. Pea type-I and type-II phosphorylase
Fig. 4.A In-vitro translation of type-I and type-II phosphorylase
isoenzymes.PolyadenylatedRNA was isolated from cotyledonsof germinating pea seeds (5 after soaking). A large-size RNA fraction was used for translation. Immunoprecipitationprocedure C was applied (see Material and methods). Lane a: precipitation with anti-type-I-isoenzymeIgG; lane b: precipitation with anti-type-IIisoenzyme IgG; lane c: preimmune IgG. CP, PP: positions of purified type-I and type-II isoenzymes.B. Immunoprecipitationof in-vitro-translated type-IIphosphorylasein the presence of purified type-I or type-II isoenzymes. PolyadenylatedRNA was isolated from cotyledonsof germinating seeds (5 d after soaking) and was translated in vitro. Immunoprecipitationwas performed(procedure C) usinganti-type-II-phosphorylaseIgG. Lanes a, e: no phosphorylase isoenzymesadded (controls); lanes b, f : 10 I~g type-II phosphorylase was added to the translation products prior to the addition of the primary antibodies; lanes c, g: l0 ~g type-I phosphorylase added. Lanes a-c : autoradiograms;lanes e-g ; protein staining with Ponceau S. Lane d: protein staining of type-I- and type-lIphosphorylase isoenzymes(3 I~geach)
isoenzymes were translated in vitro using a rabbit reticulocyte lysate. Polyadenylated RNA was isolated from cotyledons of either germinating or developing seeds. The total poly (A)+RNA preparation or a large-size mRNA fraction (obtained by sucrose density centrifugation, see Material and methods) was used for in-vitro translation. Following translation, phosphorylase isoenzymes were immunoprecipitated, separated by SDS-PAGE, transferred to nitrocellulose and, finally, were detected by autoradiography. The anti-type-II-phosphorylase IgG used for immunoprecipitation contained a low concentration of idiotypes which cross-reacted with the type-I isozyme (see Material and methods). Owing to this limited cross-reactivity the heterologous antigen (i.e. type-I phosphorylase) was, to some extent, co-precipitated. In the subsequent PAGE the co-precipitated heterologous antigen served as an internal molecular-weight marker, allowing a more precise determination of the apparent size of the type-II translation product (Fig. 4A, B). In the first experiment, poly (A)+RNA was isolated from cotyledons of germinating seeds. Immunoprecipitation with anti-type-I IgG resulted in one heavily labeled band which co-migrated with purified cytosolic phosphorylase (Fig. 4A, lane a). In a control experiment, antibodies and purified type-I phosphorylase were added simultaneously to the translation mixture. Under these conditions, the labeled protein band was completely abolished (data not shown). Thus, based upon both electrophoretic mobility and competition, the labeled band was identified as in-vitro-translated type-I phosphorylase.
J. van Berkel et al.: Glucan phosphorylasesin pea cotyledons Using anti-type-II IgG, two labeled proteins were immunoprecipitated, neither of which co-migrated with the purified homologous antigen (Fig. 4A, lane b). The faster-migrating protein was electrophoretically identical to type-I phosphorylase and represents co-precipitated heterologous antigen. The slower-migrating translation product had an apparent molecular weight of approx. 116 kDa which exceeds that of the homologous antigen by 11 kDa. For identification, immunoprecipitation was performed in the presence of either authentic type-II or type-I phosphorylase. Addition of type-II isoenzyme prevented the immunoprecipitation of the labeled 116kDa protein (Fig. 4B, lane b). Under these conditions, unlabeled type-II phosphorylase was observed in the precipitate (Fig. 4B, lane f). Thus, the 116-kDa protein is immunologically related to the type-II isoenzyme and, presumably, represents a precursor. In the competition experiment (Fig. 4B), in-vitro translation was performed using a poly(A)+RNA preparation rather than a large-size mRNA fraction. Under these conditions the immunoprecipitates consistently contained several low-molecular-weight polypeptides (Fig. 4B, lanes a-c) which were pelleted by any of the three immunoprecipitation procedures applied (see Material and methods) and with any of the antibody preparations used, including preimmune IgG. Thus, these small-size products are non-specifically precipitated. In-vitro translation of type-I phosphorylase and the precursor of type-II isoenzyme was also achieved when the reticulocyte lysate was programmed with poly (A) + RNA isolated from developing pea seeds (Fig. 5). However, the 116-kDa protein was by far more heavily labeled than type-I isoenzyme. This result was obtained by both precipitation procedures A and B. The latter, however, resulted in more non-specific precipitation of smallsize translation products (Fig. 5, lanes d-f). For in-vitro processing of the ll6-kDa protein, a stromal fraction of isolated intact chloroplasts was prepared essentially as described by Gupta and Beevers
437
Fig. 6. In-vitro processing of translated type-II phosphorylase.
PolyadenylatedRNA was isolated from cotyledonsof developing pea seeds. Translation mixtures (30 gl each) were then incubated with 100 lal buffer (lanes a, d) or with 50 gl stromal fraction (lanes b, e) or with 100 gl stromal fraction (lanes c, J). After 60 min incubationat 27~C immunoprecipitatonwas performed(procedure C) usinganti-type-II-phosphorylaseIgG. Precipitateswere detected by autoradiography(lanes a-c) or by protein stainingwithPonceau S (lanes d-f). CP, PP: positions of the purified type-I and type-II isoenzymes,pPP: position of the precursor of type-II phosophorylase. In lanes d-f, the most intensivelystained bands are IgG
(1987). Translation products were incubated for 60 min with the stromal fraction. As a control, incubation was performed (under otherwise identical conditions) in the absence of the chloroplast fraction. Following immunoprecipitation, the pelleted proteins were resolved by SDS-PAGE, transferred to nitrocellulose and were then detected by autoradiography and, for comparison, with Ponceau S (Fig. 6). After incubation with the stromal fraction, the 116kDa protein was quantitatively converted to a protein co-migrating with authentic type-II phosphorylase (Fig. 6, lanes b, c). No conversion was observed when the stromal fraction was omitted (Fig. 6, lane a). The stromal preparation contained two unlabeled type-II phosphorylase isoenzyme (cf. Fig. 1). It is interesting to note than both type-II isoenzymes were immunoprecipitated (Fig. 6, lanes e, f). Thus, the two type-II isoenzymes appear to be immunologically related.
Discussion
Fig. 5. In-vitro translation of phosphorylase isoenzymes. Poly-
adenylated RNA was isolated from cotyledonsof developing pea seeds. Immunoprecipitation was performed using procedure B (lanes a-c) or C (lanes d-f). Immunoprecipitationwas with antitype-I-phosphorylase IgG (lanes a, d) or with anti-type-IIphosphorylase IgG (lanes b, e). Lanes c, f: preimmune IgG. CP, PP: positions of purified type-I and type-II isoenzymes
Cotyledons of germinating or developing pea seeds contain both a type-I and a type-II phosphorylase. Both isoenzymes are also present in pea leaflets and in pod walls (Fig. 1). Likewise, the two enzyme forms have previously been observed in roots and etiolated shoots (Steup and Latzko 1979; Steup et al. 1986). Presumably, both proteins are expressed in any of the organs of the pea plant. In contrast, expression of an additional type-II isoenzyme in shoots appears to be controlled by the phytochrome system (Steup et al. 1986).
438 In both cotyledons (Fig. 3) and in leaflets (Conrads et al. 1986) the type-I phosphorylase resides in the cytosol of parenchyma cells. Type-II phosphorylase is a plastidspecific isoenzyme. In cotyledons o f developing seeds the type-II phosphorylase represents the main proportion of the total phosphorolytic activity. It is situated in the stromal space of starch-accumulating plastids. Although plastids of cotyledons apparently form a heterogenous organelle population (Smith et al. 1990) the enzyme was observed in plastids throughout the entire organ. In cotyledons o f germinating seeds the plastid membranes surrounding the reserve starch granules are no longer detectable. Under these conditions, the type-II isoenzyme was observed in small proplastid-like organelles. As revealed by transmission electron microscopy, these organelles possess an inner and an outer envelope membrane and contain small starch granules (data not shown). T h r o u g h o u t germination the level of type-II isoenzyme remains essentially unchanged (Fig. 2). However, a low a m o u n t o f translatable type-II m R N A appears to be present (Fig. 4). During germination, type-I phosphorylase strongly accumulates in the cytosol of parenchyma cells (Figs. 2, 3). Biosynthesis of the two compartment-specific enzyme forms was studied by in-vitro translation and processing experiments. Translation products were identified by immunoprecipitation, competition with authentic isoenzymes, and electrophoresis. Whilst type-I phosphorylase was translated as a final-size product, the apparent molecular weight of the translated type-II enzyme exceeded that of the mature protein by 11 kDa. A similar precursor size has been deduced from c D N A sequencing of type-II phosphorylase from Solanum tuberosum L. (Brisson et al. 1989). In Pisum sativum, poly(A)+RNA preparations from developing and germinating seeds resulted in the same precursor size (Fig. 4, 5). This indicates that the same precursor molecule can be imported into either amyloplasts or proplastids. Furthermore, in-vitro processing o f the precursor was achieved by incubation with a stromal fraction of chloroplasts (Fig. 6). It is, therefore, possible that chloroplasts, amyloplasts, and proplastids share a c o m m o n uptake and processing mechanism for type-II phosphorylase. Occurrence and biosynthesis o f compartment-specific phosphorylase forms in cotyledons of P. sativum, as described in this communication, differ appreciably from results recently reported for tubers of S. tuberosum. White potato tubers contain mainly type-II isoenzyme. Based on immunocytochemical studies, two reports have provided evidence for a change in compartmentation of the type-II isoenzyme during tuber maturation (Schneider et al. 1981; Brisson et al. 1989). In young tubers, the enzyme form was localized in the stromal space o f amyloplasts. However, after maturation of the tuber the enzyme was observed exclusively in the cytosol surrounding the amyloplast (which still contained an envelope membrane). Irrespective of an intra- or extraplastidic location, the enzyme was translated as a highmolecular-weight precursor. Therefore, it has been suggested that processing may occur both inside and outside the plastid (Brisson et al. 1989). In contrast, in pea plants
J. van Berkel et al.: Glucan phosphorylases in pea cotyledons (and in spinach as well; Sch~ichtele and Steup 1986) type-II phosphorylase is restricted to the plastid and a distinct cytosol-specific phosphorylase form exists which is translated immediately as a final-size product. When considering the metabolic function(s) of the plant phosphorylase forms the data presented here do not support the assumption that the plastidic phosphorylase (type II) plays a key role in starch degradation. During net starch formation a high level of this enzyme form is observed inside the amyloplasts. When massive starch mobilization occurs, type-I phosphorylase rather than type-II isoenzyme accumulates. The latter resides in proplastid-like organelles without having access to the starch granules which are degraded. Thus, it appears to be more likely that type-II phosphorylase acts on soluble glucans and catalyzes distinct steps within the complex process of starch granule formation (Viola et al. 1991). At present, the metabolic function of type-I phosphorylase is not obvious. It seems to be related to a high-molecular-weight heteroglycan which has been recently isolated from cotyledons (and shoots as well) from P. sativum (Yang and Steup 1990). This work has been made possible by grants from the Deutsche Forschungsgemeinschaft. The authors are endebted to Mrs. Karin Niehfiser for help in the immunocytochemical studies. References Anderson, D.J., Blobel, G. (1983) Immunoprecipitation of proteins from cell-free translations. Methods Enzymol. 96, 111-120 Brisson, N., Giroux, H., Zollinger, M., Camirand, A., Simard, C. (1989) Maturation and subcellular corflpartmentation of potato starch phosphorylase. Plant Cell 1, 559-566 Cashmore, A.R. (1982) The isolation of polyA + messenger RNA from higher plants. In: Methods in chloroplast molecular biology, pp. 387 392, Edelman, M., Hallick, R.B., Chua, N.-H., eds. Elsevier Biomedical Press, Amsterdam Chua, N.-H., Schmidt, G.W. (1978) Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-l,5-bisphosphate carboxylase. Proc. Natl. Acad. Sci. USA 75, 6110-6114 Conary, J., Nauerth, A., Burns, G., Hasilik, A., von Figura, K. (1986) Steroid sulfatase. Biosynthesis and processing in normal and mutant fibroblasts. Eur. J. Biochem. 158, 71-76 Conrads, J., van Berkel, J., Sch~ichtele,C., Steup, M., (1986) Nonchloroplast a-l,4-glucan phosphorylase from pea leaves: characterization and in situ localization by indirect immunofluorescence. Biochim. Biophys. Acta 882, 452-463 Gupta, S.C., Beevers, L. (1987) Regulation of nitrite reductase. Cell-free translation and processing. Plant Physiol. 83, 750-754 Hansen, S.A. (1988) Immunostaining of rocket immunoelectrophoresis precipitates too weak for identification following staining with Coomassie Brilliant Blue. Electrophoresis 9, 101-102 Hershey, H.P., Quail, P.H. (1986) Identification of cDNA clones representing phytochrome and other low abundance red-light regulated sequences. Methods Enzymol. 118, 369-383 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680-685 Nakano, K., Fukui T. (1986) The complete amino acid sequence of potato ct-glucan phosphorylase. J. Biol. Chem. 261, 8230-8236 Newgard, C.B., Hwang, P.K., Fletterick, R.J. (1989) The family of glycogen phosphorylases: structure and function. Crit. Rev. Biochem. Mol. Biol. 24, 69-98 Palm, D., Goerl, P., Burger, K.J. (1985) Evolution of catalytic and regulatory sites in phosphorylases. Nature 313, 500-502
J. van Berkel et al.: Glucan phosphorylases in pea cotyledons Rittenhouse, J., Marcus, F. (1984) Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartyl-prolyl peptide bonds in sodium dodecyl sulfate-containing buffers. Anal Biochem. 138, 442-448 Robinson, C., Ellis, R.J. (1984) Transport of proteins into chloroplasts. Partial purification of a chloroplast protease involved in the processing of imported precursor polypeptides. Eur. J. Biochem. 142, 337-342 Sch/ichtele, C., Steup, M. (1986) ct-1,4-Glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.). I. In situ localization by indirect immunofluorescence. Planta 167, 444-451 Schneider, E.M., Becker, J.-U., Volkmann, D. (1981) Biochemical properties of potato phosphorylase change with its intracellular localization as revealed by immunological methods. Planta 151, 124-134 Shimomura, S., Nagai, M., Fukui, T. (1982) Comparative glucan specificities of two types of spinach leaf phosphorylase. J. Biochem. 91, 703-717 Smith, A.M., Quinton-Tulloch, J., Denyer, K. (1990) Characteristics ofplastids responsible for starch synthesis in developing pea embryos. Planta 180, 517-523 Steup, M. (1988) Starch degradation. Biochemistry of plants, vol. 14: Carbohydrates, pp. 255-296, Preiss, J., ed. Academic Press, New York Steup, M. (1990) Starch degrading enzymes. In: Methods in plant biochemistry, pp. 103-128, Lea, P.J., ed. Academic Press, New York Steup, M., Latzko, E. (1979) Intracellular localization of phosphorylases in spinach and pea leaves. Planta 145, 69-75 Steup, M., Sch~ichtele, C. (1981) Mode of glucan degradation by
439 purified phosphorylase forms from spinach leaves. Planta 153, 351-361 Steup, M., Sch~chtele, C., Melkonian, M. (1986) Light-mediated changes in the plastidic phosphorylase patterns in shoots of Pisum sativum. Physiol. Plant. 66, 234-244 Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350M354 Viola, R., Davies, H.V., Chudek, A.R. (1991) Pathways of starch and sucrose biosynthesis in developing tubers of potato (Solanum tuberosum L.) and seeds of faba bean (Viola faba L.). Elucidation by 13C-nuclear-magnetic-resonanee spectroscopy. Planta 183, 202-208 yon Figura, K., Gieselmann, V., Hasilik, A. (1985) Mannose-6phosphate-specific receptor is a transmembrane protein with a C-terminal extension oriented towards the cytosol. Biochem. J. 225, 543-547 Westhoff, P., Zetsche, K. (1981) Regulation of the synthesis of ribulose-l,5-bisphosphate carboxylase and its subunits in the flagellate Chlorogonium elongatum. Eur. J. Biochem. 116, 261-267 Yang, Y., Steup, M. (1990) Polysaccharide fraction from higher plants which strongly interacts with the eytosolic phosphorylase isozyme. I. Isolation and characterization. Plant Physiol. 94, 960-969 Yu, F., Jen, Takeuchi, E., Inouye, M., Nakayama, H., Tagaya, M., Fukui, T. (1988) ct-Glucan phosphorylase from Escherichia coli. Cloning of the gene, and purification and characterization of the protein. J. Biol. Chem. 263, 13706-13711
P l ~ n m 9 Springer-Verlag1991
Glucan-phosphorylase forms in cotyledons of Pisum sativum L.: Localization, developmental change, in-vitro translation, and processing Joachim van Bcrkel**, Jutta Conrads-Strauch***, and Martin Steup*
Institut ffir Botanik, WestfZ,ilische Wilhelms-Universit/itMfinster, Schlossgarten 3, W-4400 Mfinster, Federal Republic of Germany Received 24 April; accepted 10 July 1991
Abstract. The occurrence, location, and biosynthesis of glucan-phosphorylase (EC 2.4.1.1) isoenzymes were studied in cotyledons of developing or germinating seeds of P i s u m s a t i v u r n L. Type-I and type-II isoenzymes were detected, and were also localized by indirect immunofluorescence using polyclonal anti-type-I or anti-type-II phosphorylase antibodies. Type-I isoenzyme was found in the cytosol of parenchyma cells whereas the type-II enzyme form is a plastid protein which resides either in amyloplasts (in developing seeds) or in proplastids (in germinating seeds). During seed development, type-II phosphorylase was the predominant isoenzyme and the type-I isoenzyme represented a very minor compound. During germination, the latter increased whilst type-II phosphorylase remained at a constant level. In in-vitro translation experiments, type-I isoenzyme was observed as a final-size product with an apparent molecular weight of approx. 90 kDa. In contrast, type-II phosphorylase was translated as a high-molecular-weight precursor (116 kDa) which, when incubated with a stromal fraction of isolated intact pea chloroplasts, was processed to the size of the mature protein (105 kDa). Key words: Glucan phosphorylase cessing - P i s u m Starch metabolism
Isoenzyme pro-
Introduction
a-l,4-Glucan phosphorylase (EC 2.4.1.1) catalyses the reaction a-D-glucose 1-phosphate+ Gn ~ ~orthophosphate + G +1 * To whom correspondence should be addressed ** Present address: Max-Planck-lnstitutf/ir Z/ichtungsforschung, Carl-von-Linn6-Weg 10, W-5000 K61n 30, Federal Republic of Germany *** Present address." John Innes Institute, Colney Lane, Norwich NR4 7UH, UK Abbreviations." IgG = immunoglobulin G; kDa = kilodalton; poly(A)+RNA = polyadenylated R N A ; SDS-PAGE= sodium dodecyl sulfate-polyacrylamidegel electrophoresis
where G and G,+ 1 represent an a-glucan composed of n and (n + 1) a-l,4-1inked glucosyl moieties, respectively. The enzyme is widely distributed in microorganisms, animals, and plants. Multiplicity is a common feature of glucan phosphorylase. In mammals, three phosphorylase isoenzymes occur (in muscle, liver, and brain) which are preferentially expressed in the respective organ and, thereby, give rise to an organ-specific pattern of phosphorylase isoenzymes (Newgard et al. 1989). Higher plants contain, within the same cell, phosphorylase isoenzymes which differ in subunit size and kinetic properties, especially in glucan specificity (Nakano and Fukui 1986; Steup 1988). One higher-plant phosphorylase (type I) has apparent monomer size of approx. 90 kDa and exhibits a relatively low affinity towards maltodextrins. Its affinity towards branched polyglucans, such as soluble starch or glycogen, is high (Steup and Schfichtele 1981; Shimomura et al. 1982). In leaves, this enzyme form resides in the cytosol of mesophyll cells (Conrads et al. 1986; Schfichtele and Steup 1986). The other phosphorylase form (type II) is restricted to the stromal space of chloroplasts (Sch/ichtele and Steup 1986), has a monomer size of more than 100 kDa, and possesses a high affinity towards maltodextrins but an extremely low affinity towards highly branched glucans, such as glycogen. In leaflets of P i s u m s a t i v u m L., two chloroplastic (type II) phosphorylase forms have been observed which are similar in size and kinetic properties (Steup and Latzko 1979; Steup et al. 1986). For the plastidic potato-tuber phosphorylase form (type-II isoenzyme), both the larger monomer size and the glucan specificity have been attributed to the existence of a unique insertion (78 amino-acid residues) which is positioned near the N-terminal/C-terminal domain junction and, possibly, impedes the binding of highly branched polysaccharides to the active site (Nakano and Fukui 1986). The metabolic implications of the dual intracellular location of higher-plant phosphorylases remain to be clarified. Two phosphorylase isoenzymes have also been observed in prokaryotes (Palm et al. 1985; Yu et al. 1988). However, both enzyme forms differ significantly from type I/type II phosphorylases of higher plants.
J. van Berkel et al.: Glucan phosphorylases in pea cotyledons I n this c o m m u n i c a t i o n , the o c c u r r e n c e a n d b i o s y n thesis o f t y p e - I a n d t y p e - I I p h o s p h o r y l a s e s h a v e been s t u d i e d in p e a c o t y l e d o n s f r o m either g r o w i n g seeds o r g e r m i n a t i n g seedlings. D e p e n d i n g u p o n the d e v e l o p m e n t a l stage, c o t y l e d o n s fulfil e n t i r e l y different m e t a bolic f u n c t i o n s : D u r i n g seed d e v e l o p m e n t , c o t y l e d o n s f u n c t i o n as a m a j o r c a r b o n sink a n d a c c u m u l a t e large q u a n t i t i e s o f starch. O n c e g e r m i n a t i o n is initiated, c o t y l e d o n s m o b i l i z e reserve s t a r c h a n d , t h e r e b y , act as the m a j o r c a r b o n source o f the entire seedling. U s i n g c o t y l e d o n s o f the v a r i o u s stages, t y p e - I a n d t y p e - I I p h o s p h o r y l a s e s were identified, quantified, a n d l o c a l i z e d in situ. T h e b i o s y n t h e s e s o f t y p e - I a n d t y p e - I I p h o s p h o r y lases were s t u d i e d b y i n - v i t r o t r a n s l a t i o n .
Material and methods Plant material Pea (Pisum sativum L. cv 'Kleine RheinlS.nderin'; Nebelung, Mfinster, FRG) seeds were surface-sterilized with sodium hypochlorite (3 % [w/v]; 60 min), washed with H20 for 4 h and were then germinated in Vermiculite under controlled conditions (Steup and Latzko 1979). Pea leaflets were harvested from 12- to 14-d-old plants grown under the same conditions. Cotyledons from developing seeds were taken from field-grown plants. Phosphorylase patterns. Extracts of pea leaflets, cotyledons or pod walls were prepared as described elsewhere (Steup 1990). Phosphorylase isoenzymes were resolved by a non-denaturing continuous electrophoresis system and were stained for glucose-l-phosphatedependent glucan-synthesizing activity as previously described (Steup 1990).
Purification of phosphorylase &oenzymes. Type-I phosphorylase was purified from pea leaflets or cotyledons (6 d after imbibition) essentially as described elsewhere (Conrads et al. 1986). Type-II isoenzyme was purified from dry seeds. The entire isolation procedure consisted of an ammonium-sulfate precipitation, ionexchange chromatography, and affinity chromatography. Dry seeds t00 g were ground to a fine powder. After the addition of 400 ml 0.1 M imidazol buffer (pH 7.0) the resulting homogenate was stirred for 15 min and centrifuged (10 min at 25 000 9). The supernatant was subjected to an ammonium-sulfate precipitation (40 to 70% saturation). The protein fraction was dissolved in 20 mM citrate buffer (pH 6.5) and dialysed against the same buffer. The sample was then applied to a diethyl aminoethyl (DEAE)-Sephacel column (5.3 cm 2 x 17.5 cm; flow 11 cm - h-1; Pharmacia, Freiburg, FRG). The column was washed with 100 ml citrate buffer and then with a linear citrate gradient ( 20 to 60 mM, pH 6.5 ; 100 ml each) to elute type-I isoenzyme (which was discarded). Type-II enzyme was eluted with a linear NaC1 gradient (0 to 0.5 M, dissolved in 60 mM citrate, pH 6.5). The enzyme preparation was then applied to an affinity gel (Sepharose-starch, deactivated with butylamine; for synthesis see Steup 1990) and eluted using a linear dextrin gradient (for details see Steup 1990). The purified type-II isoenzyme was separated from the eluents by gel filtration on Sephadex G-100 (Pharmacia; see Sch/ichtele and Steup 1986). Throughout the purification, temperature was kept at 4~ C. All media contained 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 15 gM thymol. Polyclonal antibodies. Polyclonal antibodies directed against type-I or type-II phosphorylase were raised in rabbits (Conrads et al. 1986). The anti-type-I phosphorylase immunoglobulin G (IgG) preparation did not contain idiotypes which cross-react noticeably with type-II isoenzyme (Conrads et al. 1986). Anti-type-II-phosphorylase antibodies contained a low proportion of idiotypes crossreacting with the heterologous antigen (i.e. type-I isoenzyme). These
433 idiotypes were removed from the IgG preparation as described elsewhere (Steup 1990). Immunoelectrophoresis was performed using 1% (w/v) agarose gels containing 25 mM Tricine, adjusted to pH 8.6 with Tris. Following electrophoresis (25 h at 120 V constant voltage), gels were carefully washed in 1.7% (w/v) NaCI. Precipitates were transferred to nitrocellulose and immunostained according to Hanson (1988). Indirect immunofluorescence was performed as previously described (Sch/ichtele and Steup 1986) except that the plant material was fixed in 6% (w/v) formaldehyde (overnight at room temperature) and the vacuum infiltration was omitted.
In-vitro translation and processin#. RNA was isolated from cotyledons of developing or germinating seeds according to Cashmore (1982). Polyadenylated RNA (poly(A)+RNA) was obtained by chromatography on poly U-Sepharose (Pharmacia). For size fractionation a sucrose-density-gradient centrifugation was applied (Hershey and Quail 1986). In-vitro translation was performed for 80 min at 30~ C using a rabbit reticulocyte lysate (N 90 ; Amersham, Braunschweig, FRG). Translation mixtures contained 0.1 to 0.2 lag mRNA - gl-1. For immunoprecipitation, three procedures (designated as A, B, and C) were applied. Procedure A was a modification of the technique described by Chua and Schmidt (1978) and Westhoff and Zetsche (1981). Procedure B was according to Anderson and Blobel (1983) and procedure C was according to yon Figura et al. (1985) and Conary et al. (1986). Precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE; Laemmli 1970) as modified by Rittenhouse and Marcus (1984). Proteins were transferred to nitrocellulose (Towbin et al. 1979) and were detected by autoradiography or staining with Ponceau S. For in-vitro processing a stromal fraction of isolated intact pea chloroplasts was prepared according to Robinson and Ellis (1984) and Gupta and Beevers (1987). Isolated chloroplasts were purified by centrifugation through 35% (v/v) Percoll (Pharmacia) and were broken in 20 mM Tris-HCl, pH 7.6. Proteins precipitated with ammonium sulfate (40 to 70% saturation) were dissolved in the same buffer and were desalted by filtration through Sephadex G 50 using spun columns. The protein fraction was used for processing either immediately or was stored frozen at - 70 ~ C. Processing was performed for 1 h at 27 ~ C according to Robinson and Ellis (1984) and Gupta and Beevers (1987). The processing mixture contained 30 lal translation mixture, 60 gl buffer (100 mM Hepes-KOH, pH 8.5; 220 mM KCI; 6 mM MgC12) and stromal preparation as indicated. In control mixtures, the latter was replaced by an equal volume of 20 mM Tris-HC1, pH 7.6. Phosphorylase isoenzymes were immunoprecipitated by procedure C.
Results Glucan~phosphorylase
isoenzymes
in pea
cotyledons.
Type-I and type-II phosphorylases from higher plants can be d i s t i n g u i s h e d b y affinity e l e c t r o p h o r e s i s u s i n g a g l y c o g e n - c o n t a i n i n g s e p a r a t i o n gel ( S t e u p 1990). F o r c o m p a r i s o n , e l e c t r o p h o r e s i s is p e r f o r m e d ( u n d e r o t h e r wise i d e n t i c a l c o n d i t i o n s ) in a g l u c a n - f r e e gel. O v e r a wide r a n g e o f c o n c e n t r a t i o n s , i m m o b i l i z e d g l y c o g e n d o e s n o t n o t i c e a b l y affect the m i g r a t i o n velocity o f t y p e - I I i s o e n z y m e s b u t it s t r o n g l y r e t a r d s t y p e - I enzymes: By using these criteria, t w o o f the three p h o s p h o r y l a s e s w h i c h a r e p r e s e n t in p e a leaflet e x t r a c t s were identified as t y p e - I I i s o e n z y m e s (Fig. 1A, lanes a, d). B o t h t y p e - I I e n z y m e f o r m s were o b s e r v e d in p r e p a r a t i o n s o f i s o l a t e d i n t a c t c h l o r o p l a s t s (Fig. 1A, lanes b, c). H o w e v e r , c h l o r o plasts l a c k e d the t h i r d p h o s p h o r y l a s e f o r m w h i c h is s t r o n g l y r e t a r d e d in a g l y c o g e n - c o n t a i n i n g s e p a r a t i o n gel
434
J. van Berkel et al.: Glucan phosphorylases in pea cotyledons
Fig. 2. Rocket immunoelectrophoresis of pea phosphorylase isoenzymes. Equal amounts of cotyledons of seeds 1, 3, or 6 d after soaking were homogenized in 0.1 M Tricine-Tris buffer (pH 8.6). All extracts were adjusted to the same final volume. The agarose gels contained either anti-type-l-phosphorylase IgG (left; anti-CP) or anti-type-II-phosphorylase IgG (right; anti-PP). Following electrophoresis, precipitates were transferred to nitrocellulose and were immunostained
Table 1. Type-I and type-II phosphorylase izoenzymes in extracts of cotyledons of germinating (A) or developing (B) pea seeds. Cotyledons were homogenized in 0.1 M Tricine-Tris buffer (pH 8.6). Phosphorylase isoenzymes were quantified by rocket immunoelectrophoresis. A Cotyledons were harvested 1, 3, or 6 days after soaking. B Three developmental stages were distinguished (a, b, and c) on a fresh-weight basis Fw (mg 9cotyledon - 1) Fig. 1.A Pea phosphorylase patterns. Phosphorylase isoenzymes of leaflets (lanes a, d), isolated chloroplasts (lanes b, e), cotyledons of germinating seeds (lane e: 1 d after soaking; lane f : 6 d after soaking), and pod walls (lane g) were resolved by non-denaturing continuous PAGE and subsequent activity staining. The separation gel (8.5% [w/v] total monomer concentration) was glycogen-free ( -- G) or contained 0.1% [w/v] glycogen ( + G). Approximately 70 lag protein was applied to each lane. Migration direction was from top to bottom. The arrowheads mark type-I isoenzyme which is retarded by immobilized glycogen. B Transverse gradient electrophoresis (5 to 14% (w/v) T) of extracts of pea leaflets (L) and cotyledons (C) of germinating seeds (3 d after soaking) or a 1:1 mixture of both extracts (L + C). Approximately 40 lag protein was applied to each slot. Conditions as in A
(Fig. 1A). This n o n - c h l o r o p l a s t p h o s p h o r y l a s e represents a t y p e - I i s o e n z y m e . N o n - l e a f tissues o f P i s u m satir u m c o n t a i n b o t h t y p e - I a n d t y p e - I I p h o s p h o r y l a s e s alt h o u g h their e n z y m e p a t t e r n o f t e n is less c o m p l e x t h a n t h a t o f leaflets. I n e x t r a c t s o f p e a c o t y l e d o n s two p h o s p h o r y l a s e f o r m s were r e s o l v e d b y affinity electrop h o r e s i s . O n e o f t h e m was r e t a r d e d b y i m m o b i l i z e d glyc o g e n (Fig. 1A, lanes e, f). T h e f a s t e r - m o v i n g t y p e - I I p h o s p h o r y l a s e (which is p r e s e n t in leaflets, cf. Fig. 1A, lanes a, d) was n o t d e t e c t a b l e in c o t y l e d o n s . I n p o d walls,
Isoenzyme (lag 9(mg buffer-soluble protein) - 1) Type I
Type I/ Type II
Type II
A day 1 day 3 day 6
200 220 210
0.77 2.57 7.8
2.8 2.7 3.8
0.25 0.92 2.05
60 290 700
1.5 0.7 0.3
32.8 54.3 52.1
0.04 0.01 0.006
B
stage a stage b stage c
the l a t t e r e n z y m e f o r m was o b s e r v e d as a c o m p o u n d o f m i n o r activity (Fig. 1A, lane g) w h e r e a s the m a i n b a n d s were the s a m e as t h o s e o f the c o t y l e d o n s . In a n o t h e r e x p e r i m e n t , the p h o s p h o r y l a s e f o r m s o f p e a leaflets a n d o f c o t y l e d o n s were resolved b y t r a n s v e r sal g r a d i e n t e l e c t r o p h o r e s i s using a g l y c o g e n - f r e e s e p a r a tion gel (Fig. 1B). A t a n y gel c o n c e n t r a t i o n , the t y p e - I p h o s p h o r y l a s e s o f b o t h extracts coincided. Likewise, the t y p e - I I i s o e n z y m e o f c o t y l e d o n s was e l e c t r o p h o r e t i c a l l y indiscernible f r o m the s l o w e r - m o v i n g t y p e - I I e n z y m e f o r m o f leaflets. Thus, it is highly likely t h a t the two p h o s p h o r y l a s e p r o t e i n s are identical in b o t h o r g a n s .
J. van Berkel et al.: Glucan phosphorylases in pea cotyledons
435
Fig. 3A-I. In-situ localization of type-I and type-II phosphorylase isoenzymes i cotyledons of germinating (6 d after soaking; A-F) or developing (stage b, cf. Table l ; G-I) pea seeds. Cryosections were incubated with either anti-type-Iphosphorylase IgG (D, F, I) or with anti-type-II-phosphorylase IgG (B, C, E, H) or with preimmune IgG (A, G). Magnificatons: A-D • 170; E-I, • 430. c Paraveinal tissue; A, B, D-I parenchyma cells of the cotyledons
In pea cotyledons, the ratio of type-I to type-II isoenzyme depends upon the developmental stage of the organ. The two phosphorylase forms were quantified by rocket immunoelectrophoresis followed by an electrotransfer to nitrocellulose. This modification of the conventional immunoelectrophoresis technique allows for a quantification of low-abundance proteins (Hansen 1988). Immunoelectrophoresis was performed using polyclonal anti-type-I or anti-type-II IgG. Each of the two phosphorylase isoenzymes could be monitored without interference of the heterologous antigen. This was confirmed
by control experiments in which varying amounts o f purified type-I or type-II phosphorylases were added to a crude extract o f cotyledons. The addition o f the heterologous enzyme form did not affect the immunochemical measurement o f the homologous phosphorylase protein (data not shown). In cotyledons of germinating peas, type-II phosphorylase remained at a constant level whereas the a m o u n t of type-I enzyme increased severalfold (Fig. 2). In contrast, cotyledons of developing seeds contained far more typeII phosphory!ase than type-I isoenzyme (Table 1).
436 Immunochemical localization o f phosphorylase isoenzymes. The intracellular location of type-I and type-II
phosphorylases was determined by indirect immunofluorescence. Cryosection of formaldehyde-fixed cotyledons of germinating or developing pea seeds were incubated with either anti-type-I- or anti-type-II-phosphorylase IgG (or with preimmune IgG) and, subsequently, with the fluorescein-isothiocyanate (FITC) conjugate at an appropriate dilution. Both phosphorylase isoenzymes were detected throughout the parenchyma of the cotyledons but they differed in their intracellular distribution. In cotyledons of germinating seeds, type-II phosphorylase was restricted to small proplastid-like organelles
J. van Berkel et al.: Glucan phosphorylasesin pea cotyledons which exhibited a strong fluorescence (Fig. 3b, e). These organelles were observed in the entire parenchyma tissue but they were more abundant in paraveinal cells (Fig. 3c). No fluorescence was detectable in the vicinity of the reserve starch granules. Type-I phosphorylase was observed in the entire cytosolic space of the parenchyma cells (Fig. 3d, f). In cotyledons of developing seeds, type-I phosphorylase was detected as a weak fluorescence situated in the cytosol of parenchyma cells. Neither intensity nor distribution of the immunolabel changed appreciably during seed development. Compared to type-I phosphorylase, immunolabeling of type-II isoenzyme resulted in a higher intensity of the fluorescence signal. Type-II immunolabel was restricted to the stromal space of amyloplasts. Depending upon the size of the starch granule(s) relative to that of the amyloplast, the stromal space of the organelles varied. During early stages of development, particulate starch occupied a minor proportion of the amyloplast volume and immunolabel was almost equally distributed in the entire organelle (data not shown). As starch accumulation proceeded, type-II phosphorylase isoenzyme was detected as a narrow strongly fluorescent area between the amyloplast envelope and the starch granule(s) which then occupied most of the amyloplast volume (Fig. 3h). In-vitro translation and processing o f phosphorylase isoenzymes. Pea type-I and type-II phosphorylase
Fig. 4.A In-vitro translation of type-I and type-II phosphorylase
isoenzymes.PolyadenylatedRNA was isolated from cotyledonsof germinating pea seeds (5 after soaking). A large-size RNA fraction was used for translation. Immunoprecipitationprocedure C was applied (see Material and methods). Lane a: precipitation with anti-type-I-isoenzymeIgG; lane b: precipitation with anti-type-IIisoenzyme IgG; lane c: preimmune IgG. CP, PP: positions of purified type-I and type-II isoenzymes.B. Immunoprecipitationof in-vitro-translated type-IIphosphorylasein the presence of purified type-I or type-II isoenzymes. PolyadenylatedRNA was isolated from cotyledonsof germinating seeds (5 d after soaking) and was translated in vitro. Immunoprecipitationwas performed(procedure C) usinganti-type-II-phosphorylaseIgG. Lanes a, e: no phosphorylase isoenzymesadded (controls); lanes b, f : 10 I~g type-II phosphorylase was added to the translation products prior to the addition of the primary antibodies; lanes c, g: l0 ~g type-I phosphorylase added. Lanes a-c : autoradiograms;lanes e-g ; protein staining with Ponceau S. Lane d: protein staining of type-I- and type-lIphosphorylase isoenzymes(3 I~geach)
isoenzymes were translated in vitro using a rabbit reticulocyte lysate. Polyadenylated RNA was isolated from cotyledons of either germinating or developing seeds. The total poly (A)+RNA preparation or a large-size mRNA fraction (obtained by sucrose density centrifugation, see Material and methods) was used for in-vitro translation. Following translation, phosphorylase isoenzymes were immunoprecipitated, separated by SDS-PAGE, transferred to nitrocellulose and, finally, were detected by autoradiography. The anti-type-II-phosphorylase IgG used for immunoprecipitation contained a low concentration of idiotypes which cross-reacted with the type-I isozyme (see Material and methods). Owing to this limited cross-reactivity the heterologous antigen (i.e. type-I phosphorylase) was, to some extent, co-precipitated. In the subsequent PAGE the co-precipitated heterologous antigen served as an internal molecular-weight marker, allowing a more precise determination of the apparent size of the type-II translation product (Fig. 4A, B). In the first experiment, poly (A)+RNA was isolated from cotyledons of germinating seeds. Immunoprecipitation with anti-type-I IgG resulted in one heavily labeled band which co-migrated with purified cytosolic phosphorylase (Fig. 4A, lane a). In a control experiment, antibodies and purified type-I phosphorylase were added simultaneously to the translation mixture. Under these conditions, the labeled protein band was completely abolished (data not shown). Thus, based upon both electrophoretic mobility and competition, the labeled band was identified as in-vitro-translated type-I phosphorylase.
J. van Berkel et al.: Glucan phosphorylasesin pea cotyledons Using anti-type-II IgG, two labeled proteins were immunoprecipitated, neither of which co-migrated with the purified homologous antigen (Fig. 4A, lane b). The faster-migrating protein was electrophoretically identical to type-I phosphorylase and represents co-precipitated heterologous antigen. The slower-migrating translation product had an apparent molecular weight of approx. 116 kDa which exceeds that of the homologous antigen by 11 kDa. For identification, immunoprecipitation was performed in the presence of either authentic type-II or type-I phosphorylase. Addition of type-II isoenzyme prevented the immunoprecipitation of the labeled 116kDa protein (Fig. 4B, lane b). Under these conditions, unlabeled type-II phosphorylase was observed in the precipitate (Fig. 4B, lane f). Thus, the 116-kDa protein is immunologically related to the type-II isoenzyme and, presumably, represents a precursor. In the competition experiment (Fig. 4B), in-vitro translation was performed using a poly(A)+RNA preparation rather than a large-size mRNA fraction. Under these conditions the immunoprecipitates consistently contained several low-molecular-weight polypeptides (Fig. 4B, lanes a-c) which were pelleted by any of the three immunoprecipitation procedures applied (see Material and methods) and with any of the antibody preparations used, including preimmune IgG. Thus, these small-size products are non-specifically precipitated. In-vitro translation of type-I phosphorylase and the precursor of type-II isoenzyme was also achieved when the reticulocyte lysate was programmed with poly (A) + RNA isolated from developing pea seeds (Fig. 5). However, the 116-kDa protein was by far more heavily labeled than type-I isoenzyme. This result was obtained by both precipitation procedures A and B. The latter, however, resulted in more non-specific precipitation of smallsize translation products (Fig. 5, lanes d-f). For in-vitro processing of the ll6-kDa protein, a stromal fraction of isolated intact chloroplasts was prepared essentially as described by Gupta and Beevers
437
Fig. 6. In-vitro processing of translated type-II phosphorylase.
PolyadenylatedRNA was isolated from cotyledonsof developing pea seeds. Translation mixtures (30 gl each) were then incubated with 100 lal buffer (lanes a, d) or with 50 gl stromal fraction (lanes b, e) or with 100 gl stromal fraction (lanes c, J). After 60 min incubationat 27~C immunoprecipitatonwas performed(procedure C) usinganti-type-II-phosphorylaseIgG. Precipitateswere detected by autoradiography(lanes a-c) or by protein stainingwithPonceau S (lanes d-f). CP, PP: positions of the purified type-I and type-II isoenzymes,pPP: position of the precursor of type-II phosophorylase. In lanes d-f, the most intensivelystained bands are IgG
(1987). Translation products were incubated for 60 min with the stromal fraction. As a control, incubation was performed (under otherwise identical conditions) in the absence of the chloroplast fraction. Following immunoprecipitation, the pelleted proteins were resolved by SDS-PAGE, transferred to nitrocellulose and were then detected by autoradiography and, for comparison, with Ponceau S (Fig. 6). After incubation with the stromal fraction, the 116kDa protein was quantitatively converted to a protein co-migrating with authentic type-II phosphorylase (Fig. 6, lanes b, c). No conversion was observed when the stromal fraction was omitted (Fig. 6, lane a). The stromal preparation contained two unlabeled type-II phosphorylase isoenzyme (cf. Fig. 1). It is interesting to note than both type-II isoenzymes were immunoprecipitated (Fig. 6, lanes e, f). Thus, the two type-II isoenzymes appear to be immunologically related.
Discussion
Fig. 5. In-vitro translation of phosphorylase isoenzymes. Poly-
adenylated RNA was isolated from cotyledonsof developing pea seeds. Immunoprecipitation was performed using procedure B (lanes a-c) or C (lanes d-f). Immunoprecipitationwas with antitype-I-phosphorylase IgG (lanes a, d) or with anti-type-IIphosphorylase IgG (lanes b, e). Lanes c, f: preimmune IgG. CP, PP: positions of purified type-I and type-II isoenzymes
Cotyledons of germinating or developing pea seeds contain both a type-I and a type-II phosphorylase. Both isoenzymes are also present in pea leaflets and in pod walls (Fig. 1). Likewise, the two enzyme forms have previously been observed in roots and etiolated shoots (Steup and Latzko 1979; Steup et al. 1986). Presumably, both proteins are expressed in any of the organs of the pea plant. In contrast, expression of an additional type-II isoenzyme in shoots appears to be controlled by the phytochrome system (Steup et al. 1986).
438 In both cotyledons (Fig. 3) and in leaflets (Conrads et al. 1986) the type-I phosphorylase resides in the cytosol of parenchyma cells. Type-II phosphorylase is a plastidspecific isoenzyme. In cotyledons o f developing seeds the type-II phosphorylase represents the main proportion of the total phosphorolytic activity. It is situated in the stromal space of starch-accumulating plastids. Although plastids of cotyledons apparently form a heterogenous organelle population (Smith et al. 1990) the enzyme was observed in plastids throughout the entire organ. In cotyledons o f germinating seeds the plastid membranes surrounding the reserve starch granules are no longer detectable. Under these conditions, the type-II isoenzyme was observed in small proplastid-like organelles. As revealed by transmission electron microscopy, these organelles possess an inner and an outer envelope membrane and contain small starch granules (data not shown). T h r o u g h o u t germination the level of type-II isoenzyme remains essentially unchanged (Fig. 2). However, a low a m o u n t o f translatable type-II m R N A appears to be present (Fig. 4). During germination, type-I phosphorylase strongly accumulates in the cytosol of parenchyma cells (Figs. 2, 3). Biosynthesis of the two compartment-specific enzyme forms was studied by in-vitro translation and processing experiments. Translation products were identified by immunoprecipitation, competition with authentic isoenzymes, and electrophoresis. Whilst type-I phosphorylase was translated as a final-size product, the apparent molecular weight of the translated type-II enzyme exceeded that of the mature protein by 11 kDa. A similar precursor size has been deduced from c D N A sequencing of type-II phosphorylase from Solanum tuberosum L. (Brisson et al. 1989). In Pisum sativum, poly(A)+RNA preparations from developing and germinating seeds resulted in the same precursor size (Fig. 4, 5). This indicates that the same precursor molecule can be imported into either amyloplasts or proplastids. Furthermore, in-vitro processing o f the precursor was achieved by incubation with a stromal fraction of chloroplasts (Fig. 6). It is, therefore, possible that chloroplasts, amyloplasts, and proplastids share a c o m m o n uptake and processing mechanism for type-II phosphorylase. Occurrence and biosynthesis o f compartment-specific phosphorylase forms in cotyledons of P. sativum, as described in this communication, differ appreciably from results recently reported for tubers of S. tuberosum. White potato tubers contain mainly type-II isoenzyme. Based on immunocytochemical studies, two reports have provided evidence for a change in compartmentation of the type-II isoenzyme during tuber maturation (Schneider et al. 1981; Brisson et al. 1989). In young tubers, the enzyme form was localized in the stromal space o f amyloplasts. However, after maturation of the tuber the enzyme was observed exclusively in the cytosol surrounding the amyloplast (which still contained an envelope membrane). Irrespective of an intra- or extraplastidic location, the enzyme was translated as a highmolecular-weight precursor. Therefore, it has been suggested that processing may occur both inside and outside the plastid (Brisson et al. 1989). In contrast, in pea plants
J. van Berkel et al.: Glucan phosphorylases in pea cotyledons (and in spinach as well; Sch~ichtele and Steup 1986) type-II phosphorylase is restricted to the plastid and a distinct cytosol-specific phosphorylase form exists which is translated immediately as a final-size product. When considering the metabolic function(s) of the plant phosphorylase forms the data presented here do not support the assumption that the plastidic phosphorylase (type II) plays a key role in starch degradation. During net starch formation a high level of this enzyme form is observed inside the amyloplasts. When massive starch mobilization occurs, type-I phosphorylase rather than type-II isoenzyme accumulates. The latter resides in proplastid-like organelles without having access to the starch granules which are degraded. Thus, it appears to be more likely that type-II phosphorylase acts on soluble glucans and catalyzes distinct steps within the complex process of starch granule formation (Viola et al. 1991). At present, the metabolic function of type-I phosphorylase is not obvious. It seems to be related to a high-molecular-weight heteroglycan which has been recently isolated from cotyledons (and shoots as well) from P. sativum (Yang and Steup 1990). This work has been made possible by grants from the Deutsche Forschungsgemeinschaft. The authors are endebted to Mrs. Karin Niehfiser for help in the immunocytochemical studies. References Anderson, D.J., Blobel, G. (1983) Immunoprecipitation of proteins from cell-free translations. Methods Enzymol. 96, 111-120 Brisson, N., Giroux, H., Zollinger, M., Camirand, A., Simard, C. (1989) Maturation and subcellular corflpartmentation of potato starch phosphorylase. Plant Cell 1, 559-566 Cashmore, A.R. (1982) The isolation of polyA + messenger RNA from higher plants. In: Methods in chloroplast molecular biology, pp. 387 392, Edelman, M., Hallick, R.B., Chua, N.-H., eds. Elsevier Biomedical Press, Amsterdam Chua, N.-H., Schmidt, G.W. (1978) Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-l,5-bisphosphate carboxylase. Proc. Natl. Acad. Sci. USA 75, 6110-6114 Conary, J., Nauerth, A., Burns, G., Hasilik, A., von Figura, K. (1986) Steroid sulfatase. Biosynthesis and processing in normal and mutant fibroblasts. Eur. J. Biochem. 158, 71-76 Conrads, J., van Berkel, J., Sch~ichtele,C., Steup, M., (1986) Nonchloroplast a-l,4-glucan phosphorylase from pea leaves: characterization and in situ localization by indirect immunofluorescence. Biochim. Biophys. Acta 882, 452-463 Gupta, S.C., Beevers, L. (1987) Regulation of nitrite reductase. Cell-free translation and processing. Plant Physiol. 83, 750-754 Hansen, S.A. (1988) Immunostaining of rocket immunoelectrophoresis precipitates too weak for identification following staining with Coomassie Brilliant Blue. Electrophoresis 9, 101-102 Hershey, H.P., Quail, P.H. (1986) Identification of cDNA clones representing phytochrome and other low abundance red-light regulated sequences. Methods Enzymol. 118, 369-383 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680-685 Nakano, K., Fukui T. (1986) The complete amino acid sequence of potato ct-glucan phosphorylase. J. Biol. Chem. 261, 8230-8236 Newgard, C.B., Hwang, P.K., Fletterick, R.J. (1989) The family of glycogen phosphorylases: structure and function. Crit. Rev. Biochem. Mol. Biol. 24, 69-98 Palm, D., Goerl, P., Burger, K.J. (1985) Evolution of catalytic and regulatory sites in phosphorylases. Nature 313, 500-502
J. van Berkel et al.: Glucan phosphorylases in pea cotyledons Rittenhouse, J., Marcus, F. (1984) Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartyl-prolyl peptide bonds in sodium dodecyl sulfate-containing buffers. Anal Biochem. 138, 442-448 Robinson, C., Ellis, R.J. (1984) Transport of proteins into chloroplasts. Partial purification of a chloroplast protease involved in the processing of imported precursor polypeptides. Eur. J. Biochem. 142, 337-342 Sch/ichtele, C., Steup, M. (1986) ct-1,4-Glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.). I. In situ localization by indirect immunofluorescence. Planta 167, 444-451 Schneider, E.M., Becker, J.-U., Volkmann, D. (1981) Biochemical properties of potato phosphorylase change with its intracellular localization as revealed by immunological methods. Planta 151, 124-134 Shimomura, S., Nagai, M., Fukui, T. (1982) Comparative glucan specificities of two types of spinach leaf phosphorylase. J. Biochem. 91, 703-717 Smith, A.M., Quinton-Tulloch, J., Denyer, K. (1990) Characteristics ofplastids responsible for starch synthesis in developing pea embryos. Planta 180, 517-523 Steup, M. (1988) Starch degradation. Biochemistry of plants, vol. 14: Carbohydrates, pp. 255-296, Preiss, J., ed. Academic Press, New York Steup, M. (1990) Starch degrading enzymes. In: Methods in plant biochemistry, pp. 103-128, Lea, P.J., ed. Academic Press, New York Steup, M., Latzko, E. (1979) Intracellular localization of phosphorylases in spinach and pea leaves. Planta 145, 69-75 Steup, M., Sch~ichtele, C. (1981) Mode of glucan degradation by
439 purified phosphorylase forms from spinach leaves. Planta 153, 351-361 Steup, M., Sch~chtele, C., Melkonian, M. (1986) Light-mediated changes in the plastidic phosphorylase patterns in shoots of Pisum sativum. Physiol. Plant. 66, 234-244 Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350M354 Viola, R., Davies, H.V., Chudek, A.R. (1991) Pathways of starch and sucrose biosynthesis in developing tubers of potato (Solanum tuberosum L.) and seeds of faba bean (Viola faba L.). Elucidation by 13C-nuclear-magnetic-resonanee spectroscopy. Planta 183, 202-208 yon Figura, K., Gieselmann, V., Hasilik, A. (1985) Mannose-6phosphate-specific receptor is a transmembrane protein with a C-terminal extension oriented towards the cytosol. Biochem. J. 225, 543-547 Westhoff, P., Zetsche, K. (1981) Regulation of the synthesis of ribulose-l,5-bisphosphate carboxylase and its subunits in the flagellate Chlorogonium elongatum. Eur. J. Biochem. 116, 261-267 Yang, Y., Steup, M. (1990) Polysaccharide fraction from higher plants which strongly interacts with the eytosolic phosphorylase isozyme. I. Isolation and characterization. Plant Physiol. 94, 960-969 Yu, F., Jen, Takeuchi, E., Inouye, M., Nakayama, H., Tagaya, M., Fukui, T. (1988) ct-Glucan phosphorylase from Escherichia coli. Cloning of the gene, and purification and characterization of the protein. J. Biol. Chem. 263, 13706-13711
