YEAST

VOL.

6: 33 1-343 (1990)

The Hydrophilic and Acidic N-Terminus of the Integral Membrane Enzyme Phosphatidylserine Synthase is Required for Efficient Membrane Insertion CONSTANZE SPERKA-GOTTLIEB', EVELYN-V. FASCH', KARL KUCHLER', ADAM M. BAILIS3, SUSAN A. HENRY4,FRITZ PALTAUF' AND SEPP D. KOHLWEIN'* 'Institut f i r Biochemie und Lebensmittelchemie, Technische Universitat Graz, Schlogelgasse 9/III, A 8010 Graz, Austria 'Present address: Department of Biochemistry, University of California at Berkeley, Berkeley, C A 94720, U.S.A. 'Present address: Department of Human Genetics and Development, Columbia University, College of Physicians and Surgeons, 701 West 168th Street, New York, NY 10032. U.S.A. 'Department of Biological Sciences, Carnegie MeNon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, U.S.A.

Received 9 January 1990

The product of the yeast CHOl gene, phosphatidylserine synthase (PSS), is an integral membrane protein that catalyses a central step in cellular phospholipid biosynthesis. A 1.2 kb fragment containing the regulatory and structural components of the CHOl gene was sequenced. Transcription initiation in wild-type cells was found to occur between - 1 and - 15 relative to the first ATG of a large open reading frame capable of encoding a 30 804 molecular weight protein. This translation initiation site was active in vivo and in vitro in a heterologous system. In both cases it supported production of a protein of approximately 30 000 molecular weight. A second potential translation initiation site was detected 225 or 228 bases downstream from the first ATG. This second site was active in vitro where it supported production of a protein of 22 400 molecular weight. A subclone, lacking the 5' regulatory region and the sequence encoding the first 12 amino acids of the large open reading frame, allowed translation in vivo starting at the second ATG. The resulting protein was 22 000 molecular weight, lacked the 74 N-terminal amino acids and was capable of complementing the choline auxotrophy of a chol null-mutant. In transformants carrying this construct, PSS activity and 22 kDa protein was found to be associated with membrane fractions corresponding to mitochondria and endoplasmic reticulum. However, most of the truncated PSS protein accumulated in the cytosol in an inactive form. A hybrid-protein containing the 63 N-terminal amino acids of PSS fused to mouse dihydrofolate reductase was found exclusively in the cytosol when expressed in wild-type yeast. Thus, the hydrophilic, highly acidic N-terminus of PSS is required for efficient membrane insertion but does not appear to contain sequences required for a targeting to the membrane compartment. KEY WORDS - Protein

sorting; membranes; phospholipid synthesis; yeast; Saccharomyces cerevisiae.

INTRODUCTION Most of the enzymes involved in cellular phospholipid synthesis in yeast are integral membrane proteins and have been located in mitochondria and the endoplasmic reticulum (Cobon et al., 1974; Kuchler et al., 1986). Phosphatidylserine synthase (PSS) fractionated with marker enzymes localized both to the outer mitochondria1 membrane and to the microsomal membrane compartment. However, it is not yet clear whether this protein is actually present in several subcellular compartments, or whether it is present in a non-homogeneous membrane fraction "To whom all correspondence should be addressed. 0749 503X/90/040331-13 $1 1.50 0 1990 by John Wiley & Sons Ltd

that fractionates with several membrane compartments. PSS is the first enzyme unique to the de novo pathway for the synthesis of the major membraneforming phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Studies on the susceptibility of PSS activity to several inhibitors and on its pH- and temperature-dependence demonstrated very similar properties for PSS located in both subcellular compartments (Kuchler et al., 1986).Consistent with the biochemical studies, genetic and molecular analyses identified only one structural gene, CHO1, encoding yeast PSS (Letts et al., 1983; Bailis et al., 1987; Kohlwein et al., 1988). Two different sizes have been reported for native PSS protein. Bae-Lee and Carman (1984) isolated

332 a homogeneous, active protein with an apparent molecular weight of 23 000 from microsomal membranes of glucose-grown cells. Kiyono and coworkers (Kiyono et al., 1987) have reported detection of 30 000 and 23 000 molecular weight forms of PSS. They concluded that the smaller form is the active enzyme which is produced by proteolysis of an enzymatically less active larger form. The role in vivo of the two forms of the enzyme has not been explored, however. We have isolated enzymatically active PSS from both mitochondria1 and microsoma1 membrane preparations and have observed a molecular weight of approximately 30 000 in both cases, if precaution was taken to avoid proteolysis during the isolation procedure (Kohlwein et al., 1988). It appears, therefore, that the smaller form is not the result of a specific cleavage during intracellular targeting. We have analysed the sequence of a 1.2kb fragment containing the coding and 5' regulatory regions of the CHOl gene. Two potential transcription and translation initiation sites, leading to proteins of 30 800 or 22 400 Da molecular mass, respectively, were detected. The function of the two initiation sites and the activity of the two resulting forms of PSS have been explored in vivo and in vitro. We have also analysed the partitioning of the two forms of PSS into various membrane compartments. To elucidate further the function of the Nterminal amino acids of PSS in subcellular targeting of the protein, a fusion gene containing the coding region for the N-terminal 63 amino acids of PSS fused to the structural gene encoding dihydrofolate reductase (DHFR) from mouse was constructed and expressed in yeast wild-type cells. MATERIALS AND METHODS Yeast strains and culture conditions

C. SPERKA-GOTTLIEB ETAL.

30°C. Escherichia coli strain TBI, cultivated in LB medium containing 100 mg/ml ampicillin, was used to propagate plasmids. Plasmid construction and isolation Plasmid pAB306 containing the CHOl gene on a 2.9 kb yeast genomic XhoI-Sun[ fragment (Bailis et al., 1987) in the vector pGEMITM(Promega Biotec) was digested with BamHI and PvuII. The resulting 1.9 kb fragment was inserted into the BamHI/SmaI sites of the vector pGEMITM to yield plasmid PAS 101. Subclones PAS 103 and PAS 104 were constructed by digestion of pASlOl with Hind11 and partial EcoRV digestion followed by recircularization. Subclone pAS105 was constructed by inserting the 0.9 kb EcoRI/PvuII fragment of pAB306 into the EcoRIISmaI sites of pGEMITM.Subclones pAS203 and pAS205 were constructed by insertion of the HindIIIISacI fragment of pAS103 and the HindIII/EcoRI fragment of pAS105 into the respective sites of the shuttle vector YEp352, containing the yeast URA3 gene as a selectable marker (Hill et al., 1986). Plasmid pCS303 was constructed by inserting the HindIIIIPvuII fragment of PAS 103 into the HindIII/SmaI sites of the shuttle vector YEp351, containing the yeast LEU2 gene as a selectable marker (Hill et al., 1986). Plasmid pCS343, containing the CHOI-DHFR fusion gene was constructed as follows: the 650 bp EcoRI/HindIII fragment of pDS5j2 containing the mouse DHFR gene (Stuberetal., 1984;kindlydonated by G. Schatz) was isolated, overhanging ends were filled by Klenow polymerase treatment and the resulting fragment was inserted into the EcoRV site of the CHOl gene in plasmid pCS303. YeastlE. coli shuttle vector pEH22 containing the mouse DHFR gene under the control of the yeast GAL promoter and a LEU2 selectable marker (Hurt et al., 1985) was a gift of G. Schatz. Small-scale plasmid DNA was isolated by the rapid method of Birnboim and Doly (1979). Large-scale preparations were done according to Clewell and Helinski (1969) or a protocol recommended by Promega Biotec.

Yeast strains used in this study are summarized in Table 1. Wild-type strain W303-1A was provided by R. Rothstein. Strains SDK03-IA and SDK03-IB were derived from a cross of chol null-mutant TVC1B (Bailis et al., 1987) to a wild-type strain of genotype MATuadeS (Culbertson and Henry, 1975). Media and growth conditions were as described pre- Transformation of yeast and analysis of mitotic viously (Hirsch and Henry, 1986). Transformed stability strains were cultivated on media lacking either uraYeast cells bearing the CHOI gene disruption cil or leucine, to maintain selective pressure. Media for chol null-mutants were supplemented with allele (Table 1) were transformed by the lithium 1 mmethanolamine. Auxotrophic markers were acetate method of Ito et al. (1983). Mitotic stability scored on media lacking a single component of the of the plasmids transformed into yeast was analysed complete medium. In all studies, cells were grown at by growing transformants in non-selective media for

333

HYDROPHILIC AND ACIDIC N-TERMINUS

Table 1. Yeast strains used in this study. Strain w303-IA ade5 TVC- 1B

SDK03-1A

Genotype

Remarks ‘wild-type’

MATa leu2-3,112 trpl ura3-1 his3-11.15 ade2-1 canl-100 MA Ta ade5 MA Ta chol:: TRPl trpl ura3I leu2-3.1I2 ade2-I MATa cho1::TRPI ura3-1 ade2- I

R. Rothstein

‘wild-type’

Culbertson and Henry

chol null-mutant

(1975) Bailis et al. (1987)

chol null-mutant

This study

~~

~

Transformed strain

Plasmid

Remarks

~303-IA

YEp352

SDK03-IA

YEp352

SDK03-1A

pAS203

SDK03-1A

pAS205

TVC-1B

pcs303

~ 3 0 31A -

YEp351

~303-1A

pEH22

~303-1A

pcs343

Wild-type transformedwith plasmid without CHOl insert Null-mutanttransformed with plasmid without CHOl insert Null-mutant transformedwith 1.2 kb CHOI subclone Null-mutanttransformedwith 0.9 kb CHOl subclone Null-mutant transformedwith 1.2kb CHOl subclone Wild-type transformed with vector without insert Wild-type transformed with DHFR gene Wild-type transformed with CHOI-DHFR fusion gene

more than ten generations before plating for single colonies and replica plating to selective media. Loss of prototrophy for choline and leucine or uracil (depending upon the selectable marker on the plasmid) by a significant percentage of colonies was an indication of mitotic loss of an extra-chromosomal element bearing the CHOl complementing sequence. Plasmids were retrieved from yeast by a rapid method described earlier (Hirsch and Henry, 1986).

R N A isolation and Northern blot analysis RNA was isolated from yeast by the method of Elion and Warner (1984). Polyadenylated RNA was prepared by oligo(dT)-cellulose chromatography (Aviv and Leder, 1972), fractionated on denaturi’ng formaldehyde agarose gels (Lehrach et

Source

Source This study This study This study This study This study Hill et al. (1986) and this study Hurt et al. (1985) and this study This study

al., 1977) in a modified buffer system (Rozek and Davidson, 1983), and transferred to nitrocellulose filter paper (Thomas, 1980). 32P-labelled nicktranslated pAS104-probes were synthesized as previously described (Bailis et al., 1987). Hybridization with labelled probes was carried out in 50% formamide and 0.9 M-NaCl at 37°C. 5’ end mapping The transcription start sites were identified using the Sl, nuclease mapping procedure (Berk and Sharp, 1977). Appropriate 5‘ end-labelled doublestranded D N A fragments were prepared by labelling with Y ~ ~ P - Ausing T P T4 polynucleotide kinase. Labelled fragments were recovered, purified and denatured at 80°C in the presence of 10 mg of total yeast RNA. They were allowed to form DNA/RNA

334 hybrids at 4°C for 3 h in S l hybridization buffer (80% [v/v] formamide, 40 mM-PIPES, pH 6.4, 400 mM-NaC1,2 mM-EDTA). The DNA/RNA hybrids were taken up in S1 reaction buffer (0.29 MNaCl, 50 mM-sodium acetate, pH 4.6, 4.5 mM-zinc sulfate) and exposed to 0, 10 or 20 units of nuclease Sl at 37°C for 30 min. Exposed hybrids were denatured at 95°C in sequencing gel loading buffer (95% [v/v] formamide, 2 mM-EDTA, 0.2% [w/v] bromophenol blue, 0.2% [w/v] xylene cyanol). They were subsequently flash-cooled on ice, loaded onto 8% acrylamide sequencing gels (0.5 x TBE, 8 Murea), and electrophoresed next to a sequencing ladder. The position of the protected probe relative to the sequencing ladder was visualized by autoradiography. In vitro transcription and translation The C H O l inserts in pAS103 and pASlO5 were transcribed into RNA in vitro using SP6 or T7 RNA polymerase (Boehringer-Mannheim), by the method of Melton et al. (1984) or by the protocol recommended by Promega Biotec. A cell-free proteinsynthesizing system was prepared from wheat germ using the method of Anderson er al. (1983). Translation of SP6- or T7-polymerase-generated RNA was carried out at 20°C for 60-90 min in a total volume of 100 p1 containing 40 mwpotassium acetate, 3.5 mwmagnesium acetate, 80 pwspermine, 2 mM-dithiothreitol, 12 mM-HEPES/KOH, pH 7.6, 30 ~ L each M of 19 amino acids except leucine, 50 pCi of ~-[4,5-~H]leucine (Amersham), 3 pg creatine kinase, 4 mM-ATP, 0.8 mM-GTP, 23 mwcreatine phosphate, 40 p1 wheat germ extract and 1-4 pg of SP6- or T7-polymerase-generated RNA. Addition of 10-20 pg of total bee RNA (kindly donated by G. Kreil) stimulated translation efficiency of in vitro transcribed RNA but was not essential. Labelled translation products were analysed by SDSpolyacrylamide gel electrophoresis on 12.5% slab gels (Laemmli, 1970)and visualized by fluorography (Laskey and Mills, 1975). Sequencing of CHOl DNA sequencing was performed by the method of Maxam and Gilbert (1980) with modifications for the A + G reactions of Cooke et a[. (1981). Both strands were sequenced. Preparation of subcellular fractions Microsomal membranes and mitochondria were prepared from exponentially growing wild-type and

C. SPERKA-GOTTLIEB ET AL.

transformed cells essentially as described earlier (Kuchler et al., 1986). Post-mitochondria1 supernatant was centrifuged at 18 000 x g for 15 min to remove residual mitochondria. The 18 000 x g supernatant was centrifuged for 1 h at 100 000 x g to pellet the microsomal fraction. Cytosolic fraction was a 16OOOOxg supernatant. Total membranes were prepared as described earlier (Daum et al., 1986). Cross-contamination between subcellular fractions was determined by measuring the appropriate marker enzymes as described (Kuchler et al., 1986) or by immunotitration with polyclonal antisera against porin (outer mitochondrial membrane), 40 kDa-protein (endoplasmic reticulum) and glyceraldehyde phosphate dehydrogenase (cytosol), respectively. Vacuoles were prepared from wild-type cells and null-mutants transformed with pAS205 according to Ohsumi and Anraku (198 1). a-D-Mannosidase (vacuolar membrane marker) was measured as published (Opheim, 1978). NaOH treatment of cells was performed according to Yaffe and Schatz (1984). Sodium carbonate extraction of membranes was done as described by Fujiki et al. (1982). Phosphatidylserine synthase activity in vitro and in vivo

PSS activity was assayed in isolated subcellular fractions in vitro as described earlier (Kuchler et al., 1986) by measuring the incorporation of L - [ ~ - ~ H ] serine (2 mM, 5 mCi/mol; Amersham) into chloroform/methanol2/1(v/v) extractable material. Authenticity of the labelled products was established by thin-layer chromatography using synthetic phosphatidylserine (PS) as a standard. Manganese, which is essential for activity of the enzyme, was in some cases omitted from the assay as a control, Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. PSS activity in wild-type yeast and transformants was analysed in vivo by incorporation of ~-[3-~H]serine into chloroform/methanol2/1 (v/v) extractable material. Aftera labelling period of I h a t 30"C, cells were homogenized by shaking with glass beads under C0,-cooling, and their lipids were extracted with chloroform/methanol 2/ 1 (v/v) and separated by thin-layer chromatography on Merck H-60 silicagel plates. The developing solvent was chloroform/methano1/28% ammonia, 65/35/5 (v/v/v). Identity of labelled products was established by co-chromatography with phospholipid

335

HYDROPHILIC AND ACIDIC N-TERMINUS

standards. After scraping off labelled spots, radioactivity was determined in 10 ml HP-scintillation cocktail (Beckman), containing 5% (v/v) water.

Primary structure and molecular analysis of the CHOl structural gene

The CHOl gene of yeast was isolated by complementing the choline auxotrophy of a chol mutant (Letts et al., 1983). Subsequently, it was shown that Immunohybridization CHOl encodes the mitochondrial and microsomal Subcellular fractions and whole, NaOH-treated forms of PSS (Kohlwein et aZ., 1988). The 1.2 kb cell extracts were subjected to SDS-polyacrylamide genomic DNA fragment in plasmid pAS103, which gel electrophoresis, followed by electrophoretic is capable of restoring choline prototrophy to a chol transfer to Hybond N nitrocellulose filter mutant was sequenced in both strands in its entirety (Amersham). Transfer efficiency was controlled by as described in the Methods. Parts of the DNA sePonceau S staining (Sigma, Munich). Immuno- quence and structural features of the CHOl gene decoration was carried out essentially as described and subclones of the CHOl gene used in this study (Daum et al., 1982; Haid and Suissa, 1983)using 5% are summarized in Figure 1. Kiyono et al. (1987) (w/v) non-fat dry milk in TBS (50 mM-Tris. CI pH and Nikawa et al. (1987) have also reported 8.0, 150 mM-NaC1) as blocking agent. PSS subunit sequencing of the CHOI gene. The sequence they protein was detected using rabbit antiserum raised reported is identical to the sequence presented here. against a TrpE-PSS hybrid protein purified from an We have further analysed the CHOI gene and its overexpressing E. coli strain (Kohlwein et al., 1988). transcripts as a necessary step in elucidating its Polyclonal antiserum against mouse dihydrofolate role in the overall regulation of lipid synthesis and reductase was obtained from G . Schatz. Bound the mechanisms that direct PSS to its subcellular antibody was detected by incubation with horse- destinations. radish peroxidase- or alkaline phosphataseSince the start of transcription had not been conjugated goat anti-rabbit antibodies (Accurate identified in previous studies, we determined the Chemical & Scientific Corporation, U S A . ) . sites corresponding to the 5' ends of CHOl mRNA by Sl nuclease mapping and a number of apparent transcription initiation sites were identified. The 5' end of the predominant CHOI mRNA species RESULTS AND DISCUSSION mapped to the adenine residue at - 12. The 5' ends Eukaryotic organisms contain a variety of highly of four less abundant species mapped to residues specialized membraneous structures. Most of the - 15, - 8, - 6 and - 1 (Figure 2). The ATG-codon enzymes involved in phospholipid synthesis are in- at position + 1 fits the model of Kozak (1986) for a tegral membrane proteins, and the products they preferred translation initiation site. The + 1 ATG is synthesize become structural parts of cellular mem- the start of an open reading frame of 276 amino branes (for review see Carman and Henry, 1989). acids that can be translated into a polypeptide with a Thus, in vivo compartmentation of the enzyme ac- predicted molecular weight of 30804. The size of tivities may play a role in the overall cellular control the open reading frame correlates well to the size of lipid synthesis and availability of lipids for the of native PSS (30000), which we have isolated biogenesis of cellular membranes. Synthesis of PS is from both mitochondria1 and microsomal fractions a key step in phospholipid biosynthesis in yeast. (Kohlwein et al., 1988). The 1 ATG is preceded Although this lipid per se is not essential for mito- by a TATAAATA sequence 112 bases upstream, chondrial function or cellular growth (Bailis et al., showing homology to a variety of higher eukaryotic 1987; unpublished data), it serves as a precursor for (Gannon et al., 1979) and yeast upstream sequences the major membrane-forming phospholipids, PE that have been identified as signals for transcription and PC. PSS activity has been detected in mitochon- initiation (Bennetzen and Hall, 1982; Guarente and drial and microsomal fractions in yeast wild-type Mason, 1983). A second TATAAATA sequence is cells (Cobon et al., 1974; Kuchler et al., 1986). The present at position + 150 in the CHOI gene. HowCHOl gene that encodes PSS is regulated in re- ever, there is no apparent initiation of transcription sponse to the soluble lipid precursors, inositol and from this site in vivo in wild-type cells (data not choline, in coordination with a large number of shown). The ATG codons which follow at positions enzymes of phospholipid biosynthesis (Bailis et al., +226 and +229 also fit the model of Kozak for translation initiation sites. Translation of the CHOI 1987; Carman and Henry, 1989).

+

336

C . SPERKA-GOTTLIEB ET AL. I

Aal I

ECORV

I

Xholl I

EcoRl I

I

rwhp

I

I

1

I

EcoRV 1

Ndd 1

* I

Ncol I

Pvull I

Figure 1 . Clones used in this study and detailed structural features of the CHOl gene. Plasmids were constructed as described in the Methods section. The solid line at - 163 marks a homology box to the yeast IN01 gene; the solid line between - 1 and - 15 bp upstream of the ATG marks the area of transcription starts in wild-type. P is a putative CAMP-dependent protein kinase phosphorylation site and G marks putative glycosylation sites. The numbers in brackets indicate the putatively modified amino acids.

gene starting at the first of these sites would lead to a protein with a predicted molecular weight of 22 400. Since the CHOI gene is regulated in coordination with the INOl gene which encodes inositol-lphosphate synthase, we scanned the 5' coding region of the CHOl gene for sequences shared in common with the published sequences of the INOl gene (Dean-Johnson and Henry, 1989; Carman and Henry, 1989). An inverted copy of a 9 bp element (ATGTGAAkG/,) that was found five times upstream of the INOl gene (J. Hirsch, 1987 Ph.D. thesis, Albert Einstein College of Medicine, NY; Dean-Johnson and Henry, 1989) was found at position - 163 of the CHOl gene (Figure 1). A 16 bp region of dyad symmetry reminiscent of a bacterial repressor binding site (Wharton et ai., 1984) was found at position -284. The functions of these elements with respect to CHOl and INOl gene expression are presently unknown. Overall, the amino acid sequence of the CHOI gene product, predicted from the DNA sequence, is consistent with the biochemical observations that PSS is an integral membrane protein (Bae-Lee and Carman, 1984). Two potential transmembrane helices between amino acids 86 to 108 and 150 to 190

were detected using the method of Rao and Argos (1986) as implemented in the PC/Gene analysis software package (IntelliGenetics Software, Version 6.0.1). However, the N-terminal44 amino acids are hydrophilic and highly negative in charge. A potential CAMP-dependent protein kinase phosphorylation site (Murray et al., 1984; Figure 1) at amino acid 43 and three potential glycosylation (Struck et al., 1978) sites at amino acids 60, 138 and 193, are located within the CHOl coding region. However, no glycosylation of the enzyme has been detected in vivo (G. M. Carman, personal communication). Recently, Kinney and Carman (1988) reported that the 23 000 Da form of PSS is phosphorylated by a CAMP-dependent protein kinase both in vivo and in vitro. The authors speculated that phosphorylation occurs at Ser43,since it is the only predicted CAMP-dependent protein kinase phosphorylation site in the PSS protein sequence. This potential phosphorylation site is, however, excluded in the truncated CHOl clone used in this study. As described below, the truncated clone supports production of an active 22 000 molecular weight form of PSS. The identity of the 23 000 Da-protein purified to homogeneity by Bae-Lee and Carman

337

HYDROPHILIC AND ACIDIC N-TERMINUS

A

C

G

T

AccI

EcoRI

50-

4030-

2010-

0Figure 2. SI nuclease mapping of CHOl transcripts. DNA probes were prepared by 5’ labelling pAB206 (Bailis et al., 1987) that had been linearized at the NdeI site (Figure I). A 200 bp labelled subfragment was generated by cutting with EcoRI (NdeIIEcaRI fragment). A 700 bp labelled subfragment was generated by cutting with Ace1 at a site located just to the left of the leftward EcoRV site (Figure I). The double-stranded, labelled DNA (one strand 5’ labelled) fragments were thermally denatured and renatured in the presence of 20 pg of total RNA prepared from wild-type cells. Following exposure of the DNARNA hybrids to Sl nuclease, the hybrids were denatured and run next to a sequencing ladder on 8 % acrylamide sequencing gels. A single protected species that was the same size as the original probe (not pictured) was obtained when the EcoRIINdeI fragment was used, indicating that the Send of the CHOl mRNA lies 5‘ to the EcoRI site. Five protected DNA species were observed when the AccIINdeI probe was used, all of which were shorter than the original probe. The major protected species was 50 bp longer than the single species observed when using the EcoRI/ NdeI fragment as a probe, placing the major transcriptional start site for CHOI mRNA 50 bpupstream from the EcoRI site, i.e., at - 12 on the sequence. Four minor species had lengths that corresponded to start sites at -15, -8, -6 and -1 on the CHOl sequence.

(1984) and the position of the putative phosphorylation site relative to the N-terminus need to be established. Complementation analysis of CHO 1 subclones

Plasmids pAS203 and pAS205 carrying the 1-2kb and 0.9 kb subclones of the CHOl gene on the episomal, high copy number plasmid YEp352 were transformed into chol null-mutant SDK03-1A

Figure 3. In vitro transcription and translation of CHOI subclones. SP6 or T7 RNA polymerase-generated transcripts of subclones pAS103 and pASIO5 were translated in vitro in a cell-free system from wheat germ with or without addition of bee RNA as a carrier. St: labelled molecular weight marker (67 000, 30 000, 14 100 molecular weight). Lane A: bee RNA (control); lane B: pAS103 transcript+ bee RNA; lane C: pASIO5 transcript + bee RNA; lane D pASIO5 transcript; lane E: no exogenous RNA (control). In vitro transcription and translation are described in the Methods section.

(Table 1). As shown in Figure 4, CHOl-specific transcript was absent from null-mutants transformed with the vector YEp352 without CHOl insert. Both CHOl subclones restored choline prototrophy to the null-mutant. Transformants harboring the 1.2 kb fragment overexpressed CHOI-specific message eight- to tenfold compared to wild-type cells. A transcript about 250 bases shorter than native CHOl mRNA was detected in cells transformed with the 0.9 kb CHOl subclone. Abundance of the shorter transcript was two- to threefold higher than the full-length transcript found in the wild-type control (Figure 4). Since the transcription initiation site used to produce this shortened transcript is not active in wild-type cells or in the null-mutant, produced by insertion of foreign DNA 5’ to the second site, sequences in the plasmid used for the construction of the truncated CHOl subclone may be serving to activate transcription.

338

C. SPERKA-GOTTLIEB ET AL.

Phos&tatidylserine synthase activity in vivo

A B C D E F

-1.2 Kb -0.9s Kb

Figure 4. Northern blot of poly (A)+ RNA isolated from the chol null-mutant(laneA), null-mutant transformedwithYEp352 (no insert, lane B), wild-type (lane C), null-mutant transformed withpAS205(0.9 kbCHOI insert,laneD),SP6RNApolyrnerasegenerated size standards, containing CHOl fragments: 2.9 kb, 1.8 kb, 1.2 kb (lane E) and null-mutant transformed with pAS203 (1.2 kb CHOl insert, IaneF). RNAwasisolatedfromyeastcellsas described in the Methods section, fractionated on denaturing agarose gels, blotted to nitrocellulose and hybridized with nicktranslated CHOl probe.

Activity of PSS was analysed in vivo by incorporation of L-[3- 3H]serine into PS and the phospholipids derived therefrom. Wild-type cells (W303-1A) and null-mutants transformed with different subclones of CHOI showed similar patterns of incorporation and distribution of radioactivity into lipids (Table 2). No detectable radioactivity was incorporated into lipids of the CHOl null-mutant or the null-mutant transformed with vector without an insert. Wild-type and mutant cells transformed with both the 1.2 and 0.9 kb CHOI subclones showed a similar pattern of labelled phospholipids. In both cases, label was recovered in PS, PE and PC. Thus, neither over-expression of the full-length protein or the presence of the truncated CHOl protein had a significant effect on the total or relative amounts of the phospholipids synthesized. This result is contrary to the original report that overproduction of PSS in vivo leads to overproduction of PS (Letts et al., 1983). Phosphatidylserine synthase activity in vitro and subcellular distribution

In vitro transcriptionltranslationof CHO 1 subclones Plasmids pASlO3 and PAS 105 were transcribed in vitro, and the RNAs thus generated were used to drive translation in a cell-free wheat germ lysate. Using pAS103, which contains the 1.2 kb EcoRIl PvuII full-length subclone, in vitro transcription and translation resulted in a protein of 30 000 apparent molecular weight (Figure 3). However, in some experiments, a smaller protein of about 22 000 apparent molecular weight was also observed. The smaller protein could be the result of proteolysis of the full length translation product or, alternatively, the translation process in vitro may initiate at the AUG at position +226 or +229 as well as at the initial AUG at position 1. In the in vitro system, subclone pASlO5, which contains the truncated 0.9 kb EcoRIIPvuII fragment, only supported production of the 22 000 molecular weight protein. Since this subclone lacks the information for the N-terminal 12 amino acids encoded by the intact gene, one or both of the AUG codons at position 226 or 229 pesumably serves as the translation initiation site. Thus, it appears that a second internal site in the CHOZ structural gene can serve as a translation initiation site in vitro in a heterologous system when the first AUG is eliminated.

+

+

+

Specific signal sequences are required for proper targeting of proteins to their subcellular location (Hurt and vanloon, 1986). Comparison of the primary structure of the PSS protein with other membrane proteins, however, did not reveal any common targeting sequences. The N-terminal 44 amino acids of PSS are very hydrophilic and highly acidic with a surplus of eight negatively charged amino acids, whereas signal sequences of several other yeast proteins destined for mitochondrial membranes are rather basic (for review see Hurt and vanloon, 1986). However, basic amino acids at the N-terminus are not an essential prerequisite for targeting a protein to the mitochondria, as shown for the highly acidic 17 000 molecular weight subunit of complex 111, a protein of the inner mitochondrial membrane (vanLoon et a1.,1984).No N-terminal or internal sequence homologies to outer mitochondrial proteins or proteins targeted to the endoplasmic reticulum were observed in the predicted PSS sequence. In order to analyse further the role of the hydrophilic N-terminus of PSS in subcellular targeting, we analysed the amount of protein and the specific activity of the enzyme in different subcellular fractions (Table 3). Total membranes and subcellular fractions were isolated from wild-type, mutant cells

339

HYDROPHILIC AND ACIDIC N-TERMINUS

Table 2. Incorporation of ~-[3-~H]serine into total lipids (cpm/mg dry cells), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and a non-polar lipid fraction (NPL). Total lipids (YOof wild-type) Wild-t ype SDK03-1B Null-mutant SDK03-1A Null-mutant transformed with YEp352 Null-mutant transformed with pAS203 Null-mutant transformed with pAS205

PS

PE

PC

NPL

6359

(100)

1755

2340

1494

769

21 1

(3.3)

44

59

68

60

199

(3.1)

48

48

72

32

6246

(98.2)

1624

1569

1824

1237

5793

(91.1)

1257

1610

1570

1356

Wild-type (SDK03-1B), null-mutant (SDK03-1A) and transformed null-mutants (YEp352: no insert; pAS203: 1.2 kb CHOI insert; pAS205: 0.9 kb CHOI fragment) were incubated for 1 h in the presence of ~-[3-~H]serine, lipids were extracted and analysed by thin-layer chromatography and liquid scintillation counting as described in the Methods section.

Table 3. Specific activity (nmol min-' per mg) of phosphatidylserine synthase in total membranes and subcellular fractions of wild-type (SDK03-lB), null-mutant (SDK03-I A) and null-mutants transformed with YEp352 (no insert), pAS203 (1.2 kb CHOI insert) and pAS205 (0.9 kb CHOI fragment). Total membranes With MnZ+ Without Mn2+ Wild-type SDK03-1 B Null-mutant SDK03-IA Null-mutant transformed with YEp352 Null-mutant transformed with pAS203 Null-mutant transformed with pAS205

Cytosol

Mitochondria

Microsomes

Vacuolest

12.0

0.25 (2.1%)

0-12(1.0%)

15.9

13.9

4.0

0.0

n.d.*

0.0

n.d.

n.d.

n.d.

0.0

n.d.

n.d.

n.d.

n.d.

n.d

119.2

3.61 (3.0%)

7.56 (6.3%)

62.7

65.5

n.d.

4.1

0.1 1(2.7%)

0.25 (6.1%)

4.0

3.0

0.3

*n.d.,not determined. tCells were grown on 2% glucose. Subcellular preparations and assays were performed as described in the Methods section. Cross-contamination between mitochondrial and microsomal fractions as determined by marker enzymes was below 5% and it was >lo% for the cross-contamination of vacuolar membranes with mitochondria and microsomes, respectively. Specific activities represent the mean of two independent preparations.

340

C . SPERKA-GOTTLIEB ET AL. TH

HIT PIIC CYT VAC

kD 30

W303

I

b

20

SDK03-1A/pAS205

SDK03-lA/pAS203

SDK03-1A

Figure 5. SDS-polyacrylamide gel electrophoresis and immunohybridization of total membrane preparations and subcellular fractions. Preparations of membranes and the cytosol and experimental conditions were as described in the Methods section. w303-I A is the wild-type strain transformed with vector YEp352 (no C H U l insert), SDK03-1A is a chol null-mutant, SDK03-IA/ pAS203 is the null-mutant transformed with the full-length 1.2 kb CHUZ insert in vector YEp352 (pAS203) and SDK03-IA/ pAS205 is the null-mutant transformed with the truncated 0.9 kb CHOl insert in vector YEp352 (pAS205). The amount ofprotein loaded was 25 pg with w303, 5 pg with SDK03-IA/pAS203 and 100 pg with SDK103-1A/pAS205. TM: total membranes; MIT: mitochondrial fraction (12 000 x g pellet); MIC: microsomal fraction (post-mitochondria1 100 000 x g pellet); CYT: cytosol (I60 000 x g supernatant); VAC: vacuolar fraction. Phosphatidylserine synthase was detected using antibody generated against a TRPE-PSS hybrid-protein purified from an overexpressing E. coli strain Kohlwein ef al., 1988. Bound anti-PSS antibody was visualized by incubation with horseradish peroxidaseconjugated sheep anti-rabbit antibody.

and transformants. Cross-contamination of subcellular fractions as established by assaying of marker enzymes and immunotitration was below 5%. PSS activity was about eightfold higher (as compared to wild-type cells) in membranes derived from the chol null-mutant transformed with a multicopy plasmid containing the full-length 1.2 kb subclone, pAS203. Overexpression of PSS activity corresponds well to the amount of immunoreactive protein detected in this strain (Figure 5). Null-mutants were included as a control and lacked both detectable PSS activity in vitro, as well as immunoreactive protein. The nullmutant transformed with the 0.9 kb CHOl subclone on a multicopy plasmid had PSS activity at a level of about 30% of the wild-type activity. This level of PSS activity was sufficient to restore synthesis of PS in vivo as shown in Table 2. The ratio

between mitochondrial and microsomal specific activities of PSS and the amounts of immunoreactive protein were similar in wild-type and in chol nullmutants harboring plasmids pAS203 and pAS205 (Table 3). Thus, loss of the N-terminal 74 amino acids of PSS did not change the relative distribution of the enzyme with respect to mitochondrial and microsomal membrane fractions. Furthermore, the catalytic potential of PSS was not greatly affected by deleting the N-terminus. However, omission of Mn2+ions in the in vitro assay resulted in a dramatic decrease of PSS activity in total membranes from all strains, indicating that the binding site for this ion, which is essential for enzymatic activity is not contained within the Nterminal 74 amino acids. The subcellular distribution of PSS was determined by differential centrifugation protocols and widely used marker enzymes. However, because of the lack of homogeneity of the endoplasmic reticulum, it cannot be excluded that PSS is associated with a fraction of the endoplasmic reticulum that co-fractionates with mitochondrial or other subcellular membranes. In order to resolve this issue, the marker enzymes used to localize the distribution of PSS and other membrane-associated enzymes need to be reconsidered. In null-mutants transformed with the truncated CHOI subclone, most of the PSS protein (22 000 molecular weight) accumulates in the cytosol in an inactive form (Table 3, Figure 5). Activity of PSS in the cytosol was equally low both in wild-type cells and in the transformants. Accumulation of the inactive form of the PSS protein accounts for the obvious discrepancy between the level of PSS activity and the level of overexpression of the truncated 0.9 kb CHOl transcript in chol nullmutants carrying the truncated subclone pAS205 and the specific activity of the protein observed in total membranes of this transformant. Incubation of cytosol from transformants, which contained the accumulated inactive cytosolic form of PSS, with energized mitochondria from CHOl null-mutants did not restore PSS activity or lead to incorporation of the inactive protein into membrane (data not shown). Thus, the inactive PSS protein accumulated in the cytosol is not able to insert non-specifically into membranes. PSS was not detected in any strain in the vacuole fraction. In the preparations used in these studies, vacuoles were enriched from total homogenate about 30-fold, according to the marker enzyme, a-D-mannosidase (Opheim, 1978). However, Pss specific activity and immunoreactive protein were

34 1

HYDROPHILIC AND ACIDIC N-TERMINUS

reduced in these preparations, indicating that vacuoles do not contain PSS. The very minimal PSS activity detected in our vacuole preparations was consistent with cross-contamination with mitochondria and microsomes as determined by analysis of the respective marker enzymes. Vacuolar membranes were similarly free of PSS activity and immunoreactive PSS protein in chol null-mutants transformed with plasmid pAS205, which carries the truncated C H O l construct. The fact that the native and truncated versions of PSS did not associate with vacuolar membranes argues that PSS does not simply insert non-specifically into subcellular membranes due to its hydrophobic character. Subcellular distribution of a PSS-DHFR-fusion protein

Although the proportional distribution of PSS activity was unaffected in strains expressing the 22 000 molecular weight version of PSS lacking the N-terminus, the total amount of PSS inserted into mitochondria1 and microsomal membranes was dramatically reduced. To study further the role of the N-terminus of PSS, a fusion protein containing the N-terminal 63 amino acids of PSS fused to mouse DHFR was expressed in wild-type yeast. Subcellular fractions of transformants expressing this fusion protein were isolated as described in the Methods section and subjected to immunoblotting using antiserum against mouse DHFR (Figure 6). Since the fusion protein was subject to degradation in vivo, immunoreactive protein varying in size between the full-length fusion protein and native size DHFR was detected on immunoblots of cytosolic proteins or of proteins extracted from NaOHtreated whole cells (Figure 6). PSS-DHFR fusion protein was detected exclusively in the cytosolic fraction, confirming that the N-terminus of PSS does not contain the information for targeting the protein to subcellular membranes. Conclusion

PSS, the product of the C H O l structural gene, is an integral membrane enzyme that was found to be associated with different subcellular membrane fractions. Although many studies have been carried out on targeting of proteins destined for mitochondria or for secretion outside the cell, PSS is the first protein apparently localized simultaneously to several compartments that has been analysed on a molecular level. The availability of CHOI clones

-

43

-

30

-

20

Figure 6. SDS-polyacrylamide gel electrophoresis and immunohybridization of total homogenate and subcellular fractions. Membranes and the cytosol were prepared as described in the Methods section. 20 pg of protein were loaded into each lane. Lane 1 : total homogenate of wild-type strain w303-1A transformed with vector YEp351 (no insert). Lanes 2-4: subcellular fractions of wild-type strain w303-1A transformed with plasmid pCS343, containing the coding region for the N-terminal 63 amino acids of yeast phosphatidylserine synthase (CHOI)fused to the mouse dihydrofolate reductase (DHFR)gene; lane 2: mitochondria] fraction (12 000 x gpellet); lane 3: microsomal fraction (post-mitochondria1 100 000 x g pellet); lane 4: cytosol (150 000 x g supernatant); lane 5: total homogenate of wild-type strain w303-1A transformed with plasmid pEH22 containing the structural gene encoding mouse DHFR under the control of the yeast GAL promoter. Fusion protein was detected using polyclonal antiserum against mouse DHFR (kindly provided by G. Schatz) and horseradish peroxidase-conjugated sheep antirabbit antibody.

and specific antibodies have made possible an analysis of the interaction of this protein with subcellular membranes. We have demonstrated that neither native size PSS nor its truncated form lacking the hydrophilic N-terminus, insert randomly into membranes in spite of the highly hydrophobic character of the proteins. The N-terminus of PSS, harboring mainly acidic amino acids, is not essential for enzymatic activity or targeting the protein to subcellular destinations, and does not direct a heterologous protein to subcellular membranes. The N-terminus contributes significantly, however, to efficient membrane insertion and its presence on the protein results in a higher proportion of synthesized protein finding its way into the proper membrane compartment. ACKNOWLEDGEMENTS We wish to thank Dr G. Carman for helpful discussions concerning PSS purification and

342

characterization. We wish also to thank Dr G. Kreil for providing bee mRNA, Dr G. Schatz for providing antisera and Dr J. Hill for discussion of themanuscript. This work was supported by a Schrodinger fellowship to S.D.K. and Projekt 6299 of the Fonds zur Forderung der wissenschaftlichen Forschung in Osterreich and NIH Grant GM 19629 to S.A.H. REFERENCES Anderson, C. W., Straus, J. W. and Dudock, B. S. (1 983). Preparation of a cell-free protein-synthesizing system from wheat germ. Meth. Enzymol. 101,635-644. Aviv, H. and Leder, P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. U.S.A. 69,1408-1412. Bae-Lee, M. S. and Carman, G. M. (1984). Phosphatidylserine synthesis in Saccharomyces cerevisiae. Purification and characterization of membrane-associated phosphatidylserine synthase. J . Biol. Chem. 259, 10857-10862. Bailis, A. M., Poole, M. A., Carman, G. M. and Henry, S . A. (1987). The membrane-associated enzyme phosphatidylserine synthase is regulated at the level of mRNA abundance. Mol. Cell. Biol. 7, 167-176. Bennetzen, J. L. and Hall, B. D. (1982). The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase I. J . Biol. Chem. 257,3018-3025. Berk, A. J. and Sharp, P. A. ( I 977). Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell 12,721-73 1. Birnboim, H. C. and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1 525. Carman, G. M. and Henry, S. A. (1989). Phospholipid biosynthesis in yeast. Ann. Rev. Biochem. 58,635-669. Clewell, D. B. and Helinski, D. R. (1969). Supercoiled circular DNA-protein complex in Escherichia colt Purification and induced inversion to an open circular DNA form. Proc. Natl. Acad. Sci. U.S.A. 62, 11591166. Cobon, G. S., Crowfoot, P. D. and Linnane, A. W. (1974). Biogenesis of mitochondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions. Biochem. J. 144,265-275. Cooke, N. E., Coit, D., Shine, J., Baxter, J. D. and Martial, J. A. (1981). Human prolactin. cDNA structural analysis and evolutionary comparison. J . Biol. Chem. 256,1007-1016. Culbertson, M. R. and Henry, S. A. (1975). Inositol requiring mutants of Saccharomyces cerevisiae. Genetics 80,2340. Daum, G., Bohni, P. C. and Schatz, G. (1982). Import of proteins into mitochondria. Cytochrome b, and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J. Biol. Chem. 257, 13028-1 3033.

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