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This can be achieved by adding approximately 6.67 ml of 1 M MgCI2 (commercially supplied). However, because EGTA binds Mg2+ to a small extent, a more accurate 2:3 molar ratio of Mg 2+ to EGTA over the complete series is provided when the ion is added to the CaEGTA stock at 2.01 : 3 and to the EGTA stock at 2.22: 3. The pH is then adjusted to 6.80 and the solutions brought to exactly 100 ml in volumetric flasks. 8. At this stage we have prepared two solutions, CaEGTA and EGTA, both at pH 6.80. The concentration of each is the same (nominally 100 m M ) and they can be stored for many months if they are frozen in tightly capped plastic containers. Preparing the buffers from these stocks is achieved simply by mixing them in proportions determined by the computer program, entering the concentration of free Ca 2+ required. An example is shown in Table I. Note that there will be a small displacement of Ca 2+ by added Mg 2+ and this could be compensated for, but the effect is negligible except at the lowest levels of Ca 2+. Acknowledgments This work was supported by grants from the Wellcome Trust. We thank the Royal Society and the Gower Street Secretory Mechanisms Group for funds to purchase microtiter plate readers.

[ 19] T r a n s f e r o f B u l k M a r k e r s f r o m E n d o p l a s m i c Reticulum to Plasma Membrane

By FELIX

WIELAND

Which steps connecting the sequential compartments of the endoplasmic reticulum (ER)-Golgi-plasma membrane system require special signals to determine the fate of a protein? In one view, now widely accepted and termed the "bulk flow" model, no special signal is needed to allow constitutive forward movement toward the cell surface; rather, retention signals are needed to enable resident proteins to stay in place en route, and diversion signals are needed at the trans-Golgi network for destinations other than the cell surface (for reviews, see Refs. 1- 3). Alternative views include the idea that forward movement of newly synthesized proteins is selective, requiring a special signal. 4 t R. B. Kelly, Science 230, 25 (1985). 2 S. R. Pfeffer and J. E. Rothman, Annu. Rev. Biochem. 56, 829 (1987). 3 j. E. Rothman, Cell50, 521 (1987). 4 H. F. Lodish, J. Biol. Chem. 263, 2107 (1988).

METHODS IN ENZYMOLOGY,VOL. 219

Copyright© 1992by AcademicPress,Inc. All rightsof reproductionin any formre.fred.

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In favor of the latter hypothesis is the observation that secretory and other proteins can exit the ER at widely different rates. 5-l° Major lines of evidence in favor of the bulk flow hypothesis are that prokaryotic proteins (lacking ER-to-Golgi transport signals) are secreted when redirected to the lumen of the ER, It that transplantable retention signals have now been identified for both ER membrane and luminal proteins, t2-15 and that despite 10 years of effort no definitive signal for export from the ER was found. A crucial prediction of the bulk flow hypothesis is that transport from the organdies ER and Golgi to the cell surface should occur by default. Consistent with this are the findings of retention signals in ERresident proteins (e.g., see Ref. 15) and the observations that mutations or deletions in certain Golgi resident proteins result in mislocalization to the cell surface. ~6,~7What is needed to test the bulk flow model most directly in a cell of interest is a measurement of the rate of externalization of inert compounds introduced into the lumina of the ER and Golgi cisternae in comparison to the rate of export of proteins from the same sites. Any compound present in these organdies will be secreted at some rate. The key question is kinetic competency: whether the rate of bulk flow is as fast as transit of the fastest proteins. Described here in a more general way is a concept about how to introduce small molecules as probes for the secretory pathway; also presented are luminal markers to measure the rate of transfer from the ER to the plasma membrane and from the Golgi to the plasma membrane, respectively. General Considerations An artificial luminal marker to measure the rate of vesicular transport requires the following characteristics: (1) it must be generated exclusively in the lumen of the organdie of interest; (2) it must be generated synchros T. Fitting and D. Kabat, J. Biol. Chem. 257, 14011 (1982). 6 H. F. I.xxiish, N. Kong, M. Snider, and G. J. A. M. Strous, Nature (London) 304, 80 (1983). 7 B. E. Ledford and D. F. Davis, J. Biol. Chem. 2.58, 3304 (1983). s K.-T. Yeo, J. B. Parent, T. K. Yet), and K. Olden, J. Biol. Chem. 260, 7896 0985). 9 G. Scheele and A. Tartakoff, J. Biol. Chem. 7,60, 926 0985). ~°D. B. Williams, S. J. Swiedler, andG. W. Hart,./. CellBiol. 101, 725 (1985). n M. Wiedmann, A. Huth, and T. A. Rapoport, Nature (London) 309, 637 0984). 12 S. Paabo, B. M. Bhat, W. S. M. Wold, and P. A, Perterson, Cell50, 311 (1987). ~a M. S. Poruchinsky, C. Tyndall, G. W. Both, F. Sato, A. R. Ballamy, and P. A. Atkinson, J. CellBiol. 101, 2199 (1985). ~4M. S. Poruchinsky and P. A. Atkinson, J. CellBiol. 107, 1697 (1988). ts S. Munroe and H. R. B. Pelham, Cell48, 899 (1987). ~6R. S. Fuller, A. J. Brake, and J. Thomer, Science 246, 482 (1989). 17K. J. CoUey, E. U. Lee, B. Adler, J. K. Browne, and J. C. Paulson, J. Biol. Chem. 264, 17619 (1989).

[ 19]

BULK MARKER TRANSFER FROM E R TO PLASMA MEMBRANE

191

nously in this lumen in millions of cells to allow biochemical analysis; (3) it must be trapped in the membrane system of interest so as to exclude leaking out of the cells by diffusion; (4) its generation must occur at a constant rate in the course of the experiment, i.e., its precursor must be available in excess; and (5) it must not contain any structures that could be recognized by a tentative transport receptor. Figure 1A shows this concept. A substrate (or substrate analog) that is able to diffuse through biological membranes (St) is added to the medium of cultured cells. It will penetrate all membranes and therefore equilibrate with the lumina of all organelles. However, the enzyme for this substrate is restricted to the organelle of interest, and therefore only in this lumen will the substrate be reactive.

A L

i

~S i

k2 Medium

Organelle membrane ,

r

Sd

Sd~"

Plasma membrane B

Medium content E

kl[Sd]/

0

E

Cellular content

v

Time FIG. 1. (A) Concept for the generation of a luminal marker to measure the rate of vesicular flow. (B) Evaluation of the transport rates from an experiment as explained in (A). (From Ref. 18.)

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RECONSTITUTION USING SEMIINTACT AND PERFORATED CELLS

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Here the amphilic substrate Sd is converted to yield a product (SO with hydrophilic characteristics (such as ionic charges or saccharides) that inhibit diffusion through biological membranes of this substance (Si, indiffusible substance), k t is the rate constant of formation of Si; k2 is the rate constant of vesicular transport of Si from the organelle to the plasma membrane. After expression at the plasma membrane, Si cannot remain in the outer leaflet because of its hydrophilic character and therefore is released into the medium. Evaluation of a kinetic experiment is described in Fig. 1B. Substrate Sa is added in excess and its diffusion is much faster than the subsequent reactions, so that in the cell [S] = constant. Transport follows first-order kinetics, and at steady state Si is linearly accumulated into the medium. Extrapolation of the resulting straight line to the X axis yields a lag time that represents the mean residence time of Si in the cell, z = l/k2. With first-order kinetics In 2 tl/~ =

k

t~r2 -- tin2 Therefore z is easily correlated with the half-time of transport. Practically, after addition of the labeled substrate S~ aliquots of a cell suspension are removed at various times and the content of Si in the cells and corresponding medium is determined. Extrapolation to the time axis must occur from a straight line of accumulation of S~into the medium, and as a control, the cellular level of S~during the time of linear accumulation into the medium must be constant. A luminal marker for the ER and one for the proximal Golgi will be discussed in the following section. N-Glycosylated Tripeptides as M a r k e r s for L u m e n of Endoplasmic Reticulum TM The ER is the site of N-glycosylation,19 and it has been shown in vitro that small peptides with the consensus structure X-Asn-Y-Thr/Ser- are sufficient to serve as acceptors for the unique oligosaccharide that is provided as a dolichol pyrophosphate-linked precursor. 2° We have synthesized the tripeptide Asn-Tyr-Thr and blocked its ionic charges by octanoylation of its a-amino group and by amidation of its carboxy terminus. For 18F. T. Wieland,M. L. Gleason,T. Seratini,and J. E. Rothman, Cell 50, 289 (1987). t9 R. Kornfeldand S. Kornfeld,Annu. Rev. Biochem. 54, 631 (1985). 20j. K. Welphy,P. Shenbagamurthi,W. J. Lennarz,and F. Naider,£ Biol. Chem. 258, 11856 (1983).

[19]

BULK MARKER TRANSFER FROM E R TO PLASMA MEMBRANE

193

convenient quantitation the tfipeptide was labeled with 125Iin its tyrosine residue. This compound readily diffuses into cells and is glycosylated in the ER, and the resulting glycotripeptides do not diffuse through membranes and therefore represent markers for the lumen of this organelle. Due to the minimized structure of the consensus peptide as well as the unique structure of the oligosaccharide shared by all N-glycosylated proteins in the ER, the glycopeptides are not expected to contain any signal structure that could be recognized by a putative transport receptor.

Preparation and Iodination of Tripeptide Solid-phase synthesis is performed according to Ref. 21. Benzhydrylamine-threonine-resin is used. Boc-protected amino acids as well as the resin are from Peninsula Laboratories (Belmont, CA). The peptide is cleaved from the resin by solvolysis with anhydrous hydrogen fluoride. The resultant peptide is purified by washing the resin with ether and subsequent extraction with 50% acetic acid. The dried extract is further purified by high-performance liquid chromatography (HPLC). The tripeptide H2N-Asn-Tyr-Thr-NHz elutes from an RP-I 8 column at 21% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid (TFA) in a linear gradient of 5-65% acetonitrile in 0.1% TFA at 1 ml/min. The purified peptide is acylated with octanoic acid22 using octanoic p-nitrophenyl ester. Purification is achieved by HPLC on RP-18 under conditions as described for the purification of the free tripeptide. The octanoylated peptide elutes at about 35% acetonitrile. For radioiodination, up to 50 nmol of peptide in 50 #l acetonitrile is added to 100/A of 0.5 M NaPi (pH 7.5). Between 0.5 and l0 mCi Nat2~I (carrier free; ICN Pharmaceuticals, Irvine, CA) is added. To this solution, 100/A of chloramine-T (Sigma, St. Louis, MO) (2 mg/ml) in 0.05 M NaPi (pH 7.5) is added. After 1-2 min at room temperature, the reaction is stopped by the addition of 400/tl of a solution of sodium bisulfite (2.4 mg/ml) in 0.05 M NaPi (pH 7.5). An additional 600 ~tl of water is added, and the solution is loaded onto a Sep-Pack CIa cartridge (Waters, Milford, MA) and washed first with 20 ml of 0.1% TFA and then with 20 ml of 5% acetonitrile in 0.1% TFA. The radiolabeled peptide is eluted with 60% acetonitrile in 0.1% TFA. Yields of iodination are between 25 and 50%. Specific radioactivities range from 1 × l07 tO 4 × l0 s cpm/nmol. The purity of the iodinated peptide is confirmed by HPLC (see above) and by thin-layer chromatography on silica gel plates [Si250-PA (Baker, Phillips21 j. M. Stewart and J. D. Young, Pierce Chem. Co., Rockford, Illinois, 1984. 22 M. Bodanski, "The Peptides: Analysis, Synthesis, Biology" (E. Gross and J. Meienhofer, eds.), Vol. 1, p. 106. Academic Press, New York, 1979.

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burg, NJ) in butanol/acetic acid/water (5 : 2 : 2, v/v/v)]. After drying in a Speed-Vac (Savant, Hicksville, NY) concentrator centrifuge, the radiolabeled peptides are dissolved in dimethyl sulfoxide (DMSO) at about 5 × 106 to 10 × 10s cpm//A.

Standard Assay for Quantitation of Glycosylated Tripeptide Aliquots of 200/11 are removed from cell suspensions at 0.5-1 × l 0 7 cells/ml, chilled on ice, centrifuged at 6000 g for 1 min, and the supernarant media are separated from the cell pellets. Cell pellets are extracted with 200/tl of buffer A [10 m M Tris-HCl, pH 7.4, 0.15 M NaC1, 1 m M CaCI2, 1 m M MnCI2, 0.5% (v/v) Triton X-100], and the supernatant after centrifugation in a microfuge is saved for further analysis. The media are made 0.5% (v/v) in Triton X-100, and 1 mMCaC12 and 1 m M MnCI2 are added from stock solutions. Equivalent aliquots of Triton extracts of cells and media (typically 100to 200-/d volume) are passed through small columns (200- to 400-/tl bed volume) of concanavalin A-Sepharose (Pharmacia, Piscataway, NJ) in buffer A. The columns are washed with five successive 1-ml portions of buffer A and then eluted with three 500-/d portions of 0.5 M otmethylmannoside in buffer A. 125I radioactivity in the combined o~methylmannoside eluates is determined. 125I-Labeled glycopeptides intended for further analysis (e.g., thin-layer chromatography) are prepared similarly, but after the washes with buffer A, five washes are performed with 1 ml each of buffer A without Triton X-100. Elution with t~methylmannoside is without Triton X-100 as well.

Generation of Luminal Markerfor GolgiApparatus The Golgi apparatus is known to be the site of sphingolipid biosynthesis.23,24 Sphingolipids are generated by conversion of ceramide either with phosphorylcholine (from phosphatidylcholine) to yield sphingomyelin or with glucose from UDPglucose to yield glucocerebroside. This glycolipid is further converted to a variety of higher glycosphingolipids in different cell types. We have developed an amphiphilic ceramide truncated in both the sphingosine and the fatty acyl residue to a chain length of only eight carbon atoms. This truncated ceramide (tCA) is able to diffuse through membranes and, due to its short hydrophobic chains, will not intercalate in a bilayer. Therefore the substance equilibrates into all organdies of a living 23 R. E. Pagano, Trends Biochem. Sci. 13, 202 (1988). 24 G. Van Meer, Annu. Rev. CellBiol. 5, 247 (1989).

[19]

BULK MARKER TRANSFER FROM E R TO PLASMA MEMBRANE

195

cell. In the Golgi, it is converted into a truncated sphingomyelin (tSPH) and a truncated glucocerebroside (tGlcCer). 25,u Truncated SPH turned out not to diffuse through membranes and therefore can serve as a marker for the transport of lumenal content of the Golgi apparatus. In semiintact cells tSPH transport was shown to be temperature dependent and inhibited by the unhydrolyzable GTP analog, GTPTS.27 For convenient analysis we have synthesized a radiolabeled tCA with 3H at carbon atoms 2 and 3 of its octanoyl residue.

Synthesis of D-erythro-trans-Sphingosine Cs [(2S, 3R, 4E)-2-Amino-4oct ene- l, 3-di ol] Synthesis of the truncated sphingosine is essentially as described for long-chain erythrosphingosines,2s with the following modifications: the Wittig reaction is performed using butyltriphenylphosphonium bromide, and thin-layer chromatography (TLC) of the reaction product is performed on silica gel with petroleum ether/acetic acid ethyl ester, 9:1 (Rf 0.16). The same solvent is used for analysis of the products after introduction of the azido group (Rf 0.68). Removal of the benzylidene-protecting group is performed in dry methanol without addition of dichloromethane. The products are analyzed by TLC on silica gel in dichloromethane/methanol, 95:5 (Re 0.31). Reduction of the azido group is performed in pyridine/water, 1 : 1 (rather than 2 : 1). Analysis is carded out by TLC on silica gel in chloroform/methanol, 1:1 (Rf0.19). For storage, the resulting aminocompound is transformed to its hydrochloride by acidification with methanolic HC1.

Synthesis of [~H]tCA CsCs To 100 mCi 2,3-[aH]octanoic acid (2/~mol) in 10 ml ofdichloromethane 48 mg of o-erythro-trans-spbfingosine C s hydrochloride (250/~mol) in l ml of methanol, 48/A triethylamine (500 /zmol), and 226 mg Nethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (1 mmol) are added. This 25 A. Karrenbauer, D. Jeckel, W. Just, R. Birk, R. R. Schmidt, J. E. Rothman, and F. T. Wieland, Cell 63, 259 (1990). 26 D. Jeckel, A. Karrenbauer, R. Birk, R. R. Sehmidt, and F. T. Wieland, FEBS Lett. 261, 155 (1990). 27 j. B. Helms, A. Karrenbauer, K. W. A. Wirtz, J. E. Rothman, and F. T. Wieland, J. Biol. Chem, 265, 20027 (1990). 2s p. Zimmermann and R. R. Schmidt, Liebigs Ann. Chem. p. 663 (1988).

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RECONSTITUTION USING SEMIINTACT AND PERFORATED CELLS

[ 19]

mixture is kept in a water bath and stirred at 37 °. The reaction is controlled by TLC on silica gel [chloroform/methanol/formic acid, 80: 20: 0. l (v/v/v)]. After termination of the reaction ( - 6 0 hr), the solvents are removed with a stream of nitrogen, and the yellow residue is resuspended in 0.1% TFA in water ( - 10 ml) and applied to three combined Sep-Pak C~s (Waters) cartridges, preconditioned by treatment with 80% acetonitrile and washing with 0.1% TFA in water. After washing three times with 10-ml portions of 0.1% TFA and four times with 10-ml portions of 20% acetonitrile, 0.1% TFA, the product is eluted with 20 ml of 60% acetonitrile, 0.1% TFA. One-milliliter fractions are collected. Fractions 3-16 are dried down in a Speed-vac (Savant) concentrator and the residues redissolved in 120/tl ethanol and combined. This solution is applied to an HPLC reversed-phase column (RP-18, Lichrospher, 100RP-18, 5/~m, 125 × 4 mm) and a gradient of 20 to 45% acetonitrile, 0,1% TFA in water is applied in 50 min at a flow rate of 1.0 ml/min. After 30 min, 0.25-ml fractions are collected. N-[2,3-[aH2]Octanoyl-t)-erythro-trans-sphingosineCa ([JH]CA) elutes in fractions 42-49, which are combined, dried, and resuspended in 10 ml ethanol. The yield is 34 mCi (34% of the 3H radioactivity used) and the specific radioactivity is - 50/~Ci/nmol.

StandardAssayfor QuantitationofffH]tSPH and ffH]tGlcCer Typically, 30-300/zCi of [3H]tCA together with 50-100 nmol unlabeled tCA is added per I ml of stirred Chinese hamster ovary (CHO) cells in a-modified Eagle's medium (tx-MEM) at a density of 0.5 to 1 × 107 cells/ml. Aliquots (20 to 200/d) are taken at various times, centrifuged, and the supernatant media are separated from the cell pellets. The pellets are extracted by the addition of the original volume of 50% methanol, centrifuged in a microfuge, and the supernatant is removed (cell extract). Five microliters of cell extracts and of medium is spotted onto the loading zone of a Whatman (Clifton, NJ) silica gel plate (LK6DF, 20 × 20 cm). Chromatography is performed in tanks preequilibrated with butanone/ acetone/water/formic acid, 30: 3 : 5 : 0. I (v/v). For fluorography the chromatograms are prepared according to Ref. 29. For determination of radioactivity, the chromatograms are either analyzed on a Berthold two-dimensional radioactivity scanner or radioactive spots are scraped using the corresponding fluorogram as a template, and the scraped material is analyzed by liquid scintillation counting.

29

K. Randerath, Anal Biochem. 34, 188 (1970).

[ 19]

BULK MARKER TRANSFER FROM E R TO PLASMA MEMBRANE

197

Comparison in Chinese Hamster Ovary Cells of Transport Rates from Endoplasmic Reticulum and Proximal Golgi to Plasma Membrane

Cell Culture Chinese hamster ovary wild-type cells are grown in suspension cultures in a medium (Biochrom KG) as described. The spinner bottles are kept in an incubator (5% CO2) at 37 °. For [3H]tCA incubations, cells are harvested at a density of between 5 × l05 and 8 × l05 ceUs/ml, washed with a-MEM medium, 20 m M N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES), pH 7.4, without added fetal calf serum and antibiotics, and resuspended in the same medium to give a final cell density of 0.5 × 107 to 1 X 10~ cells/ml.

A O.

>- 2 0 0 0 -

oJ

> 0 a

1000•

t.n

"1

-I

0"

'

I

41' ~ Q

.

'

0,-

I

I

~'~ 4000~

•

I

/ .~/,J~

2000

I ."

10

I

30

I

"

/

60 90 TIME (rain)

, ;

I

120

I

150

FIG. 2. Transport rates of (A) the ER marker octanoyl glycopeptides (OTP) and (B) the proximal Golgi marker truncated sphingomyelin (tSPH) in CHO cells, t8 For experimental details see text.

198

RECONSTITUTION USING SEMIINTACT AND PERFORATED CELLS

[20]

Incubation of Cells with [SH]tCA In a typical experiment, 107 CHO wild-type cells per milliliter oz-MEM (20 m M HEPES buffer, pH 7.4) is gently stirred at 37 or 30 ° in a 5% CO2 atmosphere. [3H]tCA or 125I-labeled octanoyl tripeptide (OTP) is added at time point 0 rain, and thereafter, 100-#1 aliquots are withdrawn at the time points indicated and quickly centrifuged in the cold (30 sec, 6000 g, 4°). The supernatants (media) are separated, and the pellets extracted with 100/zl 50% (v/v) methanol. After centrifugation (2 min, 10,000 g, 4 °) the supernatants are removed (cell extracts). 125I-I:abeled glycotripeptides and tSPH are determined as described above. The result is shown in Fig. 2. At 30 °, the proximal Golgi marker tSPH is transported to the cell surface with a lag time of 20 min, whereas the lag time of transport of the ER markers is about 28 min. This corresponds to half-times of transport of about 14 and 21 min, respectively. Thus, the half-time of transport from the ER to the proximal Golgi can be estimated to be 7 rain at 30 °. The bulk transport rates measured with the two markers presented here indicate that no special signal-receptor system is involved in the secretion of proteins from the ER in the Golgi apparatus, but rather the proteins travel with the bulk of the material, and proteins destined for individual stations of the pathway must be sorted out from the unsignaled flow. 3° 30 H. R. B. Pelham,

Annu. Rev. CellBiol. 5,

1 (1989).

[20] R e c o n s t i t u t i o n o f E n d o c y t o s i s a n d R e c y c l i n g U s i n g Perforated Madin-Darby Canine Kidney Cells By

BENJAMIN PODBILIEWICZ

and IRA MEU.MAN

Endocytosis in animal cells is characterized by the internalization of extracellular fluid and macromolecular ligands bound to cell surface receptors. In general, endocytosis involves the formation of small (0.10.2 gm) transport vesicles that form from the invagination and pinching off of dathrin-coated pits of the plasma membrane. While some toxins and lectins appear to be internalized in uncoated vesicles, in many cells, it is apparent that the formation of coated vesicles represents the quantitatively most important route of entry into the cell. After the formation, coated vesicles uncoat and fuse with acidic early endosomes (pH 6.0-6.3) within minutes. Often, but not always, ligands METHODS IN ENZYMOLOGY, VOL. 219

Copyright© 1992by AcademicPress,Inc. All rightsofreproduc~onin any form reservecL