A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast

Cell, Vol. 53, 753-768, June 3, 1988, CopyrIght 0 1988 by Cell Press

A GTP-Binding Protein Required for Secretion Rapidly Associates with Secretory Vesicles and the PI&ma Membrane in Yeast Bruno Goud, Antti Salminen, and Peter J. Novick

Nancy C. Walworth,

Department of Cell Biology Yale University School of Medicine 333 Cedar Street, P. 0. Box 3333 New Haven, Connecticut 06510

Summary SEW, one of the 10 genes involved in the final stage of the yeast secretory pathway, encodes a ras-like, GTP-binding protein. In wild-type cells, Sec4 protein is located on the cytoplasmic face of both the plasma membrane and the secretory vesicles in transit to the cell surface. In all post-Golgi blocked set mutants, Sec4p is predominantly associated with the secretory vesicles that accumulate as a result of the secretory block. Sec4p is synthesized as a soluble protein that rapidly (fljZ < 1 min) and tightly associates with secretory vesicles and the plasma membrane by virtue of a conformational change or a covalent modification. These data suggest that Sec4p may function as a “G” protein on the vesicle surface to transduce an intracellular signal needed to regulate transport between the Golgi apparatus and the plasma membrane.

Introduction Transport of newly synthesized proteins from the endoplasmic reticulum (Et?) to the cell surface or to the extracellular space is accomplished by the well-defined, exocytic pathway (Palade, 1975). At each stage of this pathway, transport involves the movement of carrier vesicles. Vesicles bud from the ER and fuse with the cis aspect of the Golgi complex. Passage through the various stacks of the Golgi complex is also mediated by vesicular traffic. Fusion of Golgi-derived vesicles with the plasma membrane completes the protein export pathway. Despite the generality of this mechanism, the principles governing the flow of vesicular traffic remain poorly understood at a molecular level. The yeast Saccharomyces cerevisiae, in which many of the features of mammalian protein secretion are conserved (Novick et al., 1981), offers powerful tools to address these mechanisms. Genetic analysis of the yeast exocytic pathway has defined 25 genes whose products are essential for protein transport from the ER to the cell surface (Novick and Schekman, 1979; Novick et al., 1980; Newman and Ferro-Novick, 1987). Among them, ten genes govern the transport between the Golgi apparatus and the plasma membrane. Thermosensitive alleles of these genes have been isolated. At their restrictive temperature, these mutants are blocked in the transport of all cell surface proteins (Novick and Schekman, 1983), and as a result, postGolgi secretory vesicles accumulate. We have recently cloned and sequenced one of these late-acting genes, SEC4 (Salminen and Novick, 1987).

This gene encodes a ras-like protein with a predicted molecular weight of 23.5 kd. The regions of the ras protein sequence most similar to the Sec4 protein (Sec4p) are the four domains thought to serve collectively as a GTPbinding site (McCormick et al., 1985; Jurnak, 1985). Genetic analysis has shown that strong interactions exist between the SEC4 gene and three other late-acting SEC genes, suggesting that Sec4p may act as a mnster regulator of the final stage of the yeast secretory pathway. The finding that a putative GTP-binding protein is involved in the control of exocytosis in yeast is not entirely unexpected. Indeed, there is growing evidence that GTP analogs can directly activate the exocytic mechanism in some secretory cells such as neutrophils or mast cells (Fernandez et al., 1984; Barrowman et al., 1986; Howell et al., 1987). GTP-binding proteins have also been recently implicated in the regulation of vesicular transport from the ER to the Golgi complex (Segev et al., 1988) and transport from the cis stack of the Golgi complex to the medial stack (MelanGon et al., 1987). Finally, Bar-Sagi and Feramisco (1986) have shown that the microinjection of p21 ras protein into fibroblasts leads to an increase in membrane ruffling and fluid-phase endocytosis. This experiment suggests a new role for ras proteins in the regulation of membrane traffic. The above data suggest that a set of GTP-binding proteins may control the various steps of the exocytic pathway. Each class of carrier vesicles could possess its own, distinct GTP-binding protein. It is therefore of interest to characterize and localize the Sec4p. In this report, we show evidence that the SEC4 gene encodes a GTPbinding protein that rapidly associates with the cytoplasmic surface of secretory vesicles as well as the inner surface of the plasma membrane, and we explore the biochemical nature of this interaction. Our findings suggest that Sec4p may primarily act on the surface of secrletory vesicles to directly transduce an intracellular signal needed to regulate transport between the Golgi apparatus and the plasma membrane.

Results Sec4p Is a GTP-Binding

Protein

As shown in our previous paper (Salminen and Novick, 1987), the sequence of the SEC4 gene predicts a protein product of about 23.5 kd molecular weight. We have raised polyclonal rabbit antibodies against a TrpE-Sec4 fusion protein (see Experimental Procedures). By Western blot analysis, these antibodies recognized a protein in a wildtype yeast lysate that migrated at the expected molecular weight (Figure 1A). To establish that this protein represents the product of the SEC4 gene, we used ?he strong, inducible GAL7 promoter to overproduce Sec4.p. A wild-type, Gal+ strain was transformed with the G,4L7-SEC4 gene fusion plasmid, pNB219. This plasmid was integrated into the SEC4 locus creating aduplication of the gene in which one copy is under GAL7 control and is induced when cells

Cell 754

A. Sec4p Immunoblot

GAL~~4-8 1

B. 32P-GTP Binding

9~4-8

WT

GALSEC4

2

3

4

GAL~~4.8 1

~~4.8

W-T

GALSEC4

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3

4

/Cell Fractionation

Scheme\

at 25:

in 2% glucose

Grow cells

& Shift to 37q In 0.2% glucose

Spheroplast

6 In I 4M sorbitol

4 Lyse cells rn 08M sorbitol,

dounce

Very low speed spin,

450 G, 3 mn

Sl Spin

Figure 1. Sec4p Is a GTP-Binding

IOKG,

10 ml”

Protein

GAL Sec4-8 refers to a strain (NY499) in which the temperaturesensitive form of the protein is expressed behind the GAL1 promoter; Sec4-8 refers to the original temperature-sensitive strain (NY405); WT refers to the wild-type strain (NY13); GAL Sec4 refers to a strain in which the normal Sec4 protein is expressed behind the GAL7 promoter (NY494). (A) Wild-type and Sec4-8 cells were grown in YP 2% glucose. GAL Sec4-8 and GAL Sec4 cells were induced for 3 hr in YP 2% galactose. Cells were then broken by agitation with glass beads and boiled in SDS. Lysates were run on a 12% SDS-PAGE gel and the gel was transferred to nitrocellulose. The filter was probed with the anti-Sec4p antibody (dilution l/1000) and a goat anti-rabbit IgG alkaline phosphatase conjugate. (6) A parallel series of lanes were loaded and transferred to nitrocellulose. This blot was incubated with [~J-~“P]GTP. Five GTP-binding proteins are revealed by this procedure. The middle band co-migrates with the Sec4p as identified in wild-type cells with the anti-Sec4p antibody (A). This GTP-binding band is overproduced when the Sec4p is expressed behind a strong promoter. The Sec4-8 mutant protein is slightly shifted in mobility and fails to bind GTP, even when overproduced from the GAL1 promoter.

are shifted to galactose. lmmunoblot analysis of lysates derived from wild-type and plasmid-containing cells showed a specific, galactose-induced increase of the 23.5 kd immunoreactive protein in the plasmid-containing cells (Figure 1A). This indicates that the antibody indeed recognizes the Sec4p. The se&-8 allele encodes a protein containing a substitution of glycine to aspartate at amino acid 147 (Salminen and Novick, 1987). By Western blot, the immunoreactive mutant protein was less abundant in sec#-8 than in wild-type lysates and was shifted to a slightly lower mobility. This shifted band was overproduced when the sec4-8 coding sequence was placed behind the GAL7 promoter (Figure lA), confirming the specificity of the antibody. We have shown in our previous study that among known protein sequences the strongest similarity is between Sec4p and another yeast ras homolog, the YPTl gene product (Gallwitz et al., 1983). Sec4p shares 47.5% se-

Sephacryl

S-l 000 column

Figure 2. Cell Fractionation

Scheme

quence identity with this protein, yet the predicted size of Sec4p is about 1 kd larger than Yptlp. Although the results presented above indicate that the antibody recognizes Sec4p, we determined if it would also recognize Yptlp. To investigate this possibility, we performed immunoblots on lysates obtained from wild-type, Sec4p overproducing and Yptlp overproducing cells. Duplicate blots were probed with either anti-Sec4p or anti-Yptlp antibodies (a gift of Dr. Nava Segev), and two distinct proteins were identified, consistent with the fact that Sec4p and Yptlp are separated on a 10% SDS gel and that there is no crossreactivity between the two antibodies. The mobilities of these proteins correspond to the sizes predicted by their sequences (data not shown). The most conserved regions between ras proteins and Sec4p are those implicated in GTP-binding (Salminen and Novick, 1987). We have directly tested the GTP-binding activity of Sec4p. Previous studies have shown that some GTP-binding proteins can renature following SDS gel electrophoresis and transfer to nitrocellulose (McGrath et al., 1984; Schmitt et al., 1986; Lapetina and Reep, 1987). To utilize this technique, total protein from a wild-type strain and Sec4p overproducing strain was separated by SDS gel electrophoresis and transferred to a nitrocellulose filter. The filter was incubated in the presence of a-32Plabeled GTF’. Autoradiography of the filter revealed a distinct pattern of GTP-binding proteins (Figure 1B). The middle band co-migrated with the Sec4p, as demonstrated in

Localization

of Sec4 Protein

755

Figure 3. Distrrbution of the Sec4p from WildType and sec6-4 Cells during Differential Centrifugation

wt

Sl

lC0Kg

1w

45og Pl

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Wild-type (NY13) and se&4 (NY17) cells were grown at 25% in YP 2% glucose and shifted to 37°C for 2 hr in YP medium containing 0.2% glucose. The cells were then spheroplasted and lysed osmotically. The lysates were spun at 450 x g to generate Pl and Sl. Sl was spun at 10,000 x g to generate P2 and S2. S2 was spun at 100,000 x g for 1 hr to generate P3 and S3. Equal aliquots of all samples were boiled in SDS and run on a 10% SDS gel. The gel was transferred to nitroceilulose and the Sec4p was detected by a quantitative Western blot protocol usrng ‘251-Protern A. The quantitation is shown in the bottom panel. The results are expressed as the percentage of cpm found in unfractionated lysates. A shift in the distribution of Sec4p from P2 to P3 is seen in the vesicle accumulating mutant at the restrictive temperature.

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a parallel immunoblot with the antiSec4p antibody. The signal of this band was amplified more than lo-fold with the Sec4p overproducer, indicating that Sec4p is a GTPbinding protein In the case of the se&-8 strain, which carries the temperature-sensitive allele of SEC4, the GTP-binding band was absent (Figure 16). While the total amount of the Sec4-8 protein was decreased by at least five times as compared with wild-type cells, overproduction of the Sec4-8 protein by expression from the GAL.7 promoter did not restore this GTP-binding band. The specificity of GTP-binding by Sec4p was demonstrated in a competition binding experiment. Filters were incubated in the presence of cold ATP or GTP (20, 200, or 2000 times molar excess) prior to the addition of [32P]GTP to the reaction buffer. A large excess of ATP failed to compete with [32P]GTP in this binding assay, whereas the presence of a 20-fold molar excess of cold GTP was sufficient to abolish the apparent binding of labeled nucleotide (data not shown).

Sec4p Is Located on the Plasma Membrane and Secretory Vesicles The intracellular location of Sec4p has been studied in wild-type cells and in a vesicle accumulating mutant, sec6-4. For this purpose, we developed the fractionation orotocol outlined in Figure 2. Cells were grown at 25% in rich medium containing only 0.2% glucose and then transferred to the restrictive temperature (37%) in medium containing 2% glucose. The shift to 37°C causes sec6-4 cells to accumulate post-Golgi secretory vesicles (Novick et al., l981), while the decrease in glucose concentration allows derepression of invertase biosynthesis. Thus, invertase can be used as a lumenal marker of the accumulated vesi-

cles. After 2 hr, the cells were converted to spheroplasts and lysed osmotically. The lysis buffer used has been shown to stabilize the yeast vacuole and the secretory vesicles (Markarow, 1985; Walworth and Novick, 1987). The lysates were spun at 450 x g to remove unlysed cells, and the supernatants (Sl) were then successively centrifuged at 10,000 x g and 100,000 x g. Aliquots of the different supernatants (Sl, S2, S3) and pellets (PI, P2, P3) generated during the differential centrifugation were subjected to SDS gel electrophoresis and transferred onto nitrocellulose. The Sec4p was revealed and quantitated using the anti-Sec4p antibody and ‘*sl-Protein A (Figure 3). In these experiments, Sec4p appeared as a doublet, which was especially prominent in P3 pellets (Figure 3, upper panel). The fact that we observed only the upper band of thris doublet when cells were broken with glass beads and immediately boiled in SDS (see for example Figure 1 and Figure 9) indicates that the appearance of the lower band is probably due to proteolytic cleavage of Sec4p during differential centrifugation. The total amount of Sec4p was similar in wild-type and sec6-4 cells. As can be seen from the quantitative fractionation data presented in Figure 3, lower panel, most of the Sec4p was associated with either the P2 or P3 pellets, which indicates that the Sec4p is largely membranebound in both strains. However, the distribution of Sec4p differed in these two strains: in wild-type, Sec4p was predominantly associated with the P2 pell’et, whereas, in sec6-4 cells, the microsomal pellet (P3) contained about 50% of the total Sec4p. The distribution of various enzyme markers was also monitored in these two pellets. P2 contained 60% of the total NADPH-cytochrome c reductase enzyme activity found in the initial lysate. This enzyme is a marker of ER membrane (Kreibich et at., 1973; Kubota

Cell 756

+.

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03 02

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01 0.0

i E f”

6o

ES 22

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s

*O

6 4

EE x

with the Plasma Membrane

200

0 0

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fractions

frsctions Figure 4. Sec4p Comigrates

2

Enzyme Marker rn P2 Pellets of Wild-Type (A) and sec6-4 Cells (8)

The P2 pellets were generated by differential centrifugation and loaded in 60% sucrose (2 ml) on the bottom of 35%-60% sucrose gradienrs. After centrifugation to equilibrium, the gradients were fractionated from the top (fraction 1) in 21 fractions (0.6 ml) and the residual pellets were resuspended in 0.6 ml of 60% sucrose (fraction 22). In each fraction, various marker enzymes were assayed and the amount of Sec4p was determined by quantitative Western blot analysis. Results are expressed as the total activity per fraction. Density (g/ml); proteins (ng); NADPH-cytochrome c reductase (ttmoles of cytochrome c reduced per min); Cytochrome c oxidase (nmoles of cytochrome c oxidized per min); a-mannosidase (nmoles of p-nitrophenyl-u-D-manno-pyranoside hydrolyzed per hr); invertase (r.lM of glucose produced per min); vanadate-sensitive Mg’+-ATPase (nmoles of phosphate produced per min); Sec4p (total cpm).

et al., 1976). P2 also contained 70% of the mitochondrial inner membrane enzyme marker enzyme cytochrome c oxidase (Mason et al., 1973). The plasma membrane enzyme marker, vanadate-sensitive Mg2+ ATPase (Bowman and Slayman, 1979; Willsky, 1979) was also enriched in P2 pellets. Only very small amounts of ER, mitochondrial, and plasma membrane marker enzyme activities were found in P3 pellets (respectively, 40/o, 3010,and 1% of the total activity in the wild-type P3 fraction and 7%, 40/o, and 4% of the total activity in the sec6-4 P3 fraction). To determine the distribution of secretory vesicles in the sec6-4 cell fractions, invertase activity was measured in the presence and absence of Triton X-100. More than 70% of the intravesicular invertase (active in the presence of Triton X-100, but not in its absence) was recovered in P3, in agreement with previous experiments showing that secretory vesicles accumulated in sec6-4 cells are greatly enriched in the microsomal pellet (Walworth and Novick, 1987). To determine with which subcellular compartment(s) Sec4p was associated, P2 and P3 pellets from both strains

were subfractionated on sucrose density gradients. Each fraction was analyzed for the different enzyme markers and the amount of Sec4p quantitated. As can be seen in Figure 4A, the Sec4p present in P2 pellets from wild-type cells co-migrated with the plasma membrane enzyme marker, vanadate-sensitive ATPase. This peak of plasma membrane activity is lo-fold purified with respect to the Sl fraction as determined by the increase in the specific activity of the vanadate-sensitive ATPase. This marker was at least partially resolved from mitochondrial and ER membrane markers as well as from a vacuole marker, a-mannosidase. An identical result was obtained utilizing a P2 pellet from sec6-4 cells (Figure 4B), except that the total amount of Sec4p was about 5-fold reduced compared with wild-type P2. It should be noted that the invertase activity (about 10% of the total invertase activity is present in this pellet) was not located at the same position on the gradient as the peak of Sec4p. The Sec4p present in the P3 pellets from either strain banded at a higher density on the sucrose gradients (Figure 5) than did the P2 Sec4p pools. In the case of the sec6-

Localization 757

of Sec4 Protein

Figure 5. Fractionation of P3 Pellets from WildType (A) and se&-4 Ceils (B) on Sucrose Gradients The P3 pellets were gen’erated by differential centrifugation and loadeNd (2 ml) in 60% sucrose on the bottom of 35%60% sucrose gradients The presence of Sec4p was monitored and the invertase activity was measured as described in Figure 4. The invertase activity comigrates with Sec4p in se&# cells, In wildtype cells, Sec4p peaks at the same density as in se&-4 cells.

sect%4

d

5

10

15

20

25

fractions

4 P3 gradient, the Sec4p co-fractionated with invertase activity marking the secretory vesicles (Figure 5B). The Sec4p present in wild-type P3 (Figure 5A) was five times less abundant, but peaked at the same high density, suggesting that it too may be associated with secretory vesicles. To further characterize the microsomal pool of Sec4p, we have used an alternate fractionation procedure, specifically designed to yield pure secretory vesicles (Walworth and Novick, 1987). Frgure 6 shows the fractionation of P3 pellets from se&-4 and wild-type cells on a Sephacryl S-1000 column. On the column of sec6-4 microsomes (Figure 6A), Sec4p co-eluted with invertase activity as predicted. We have previously shown, by several techniques, that this region of the sec6-4 elution profile contains secretory vesicles that are at least 80% pure (Walworth and Novick, 1987). Although we have not followed a Golgi marker enzyme, the identical fractionation protocol has been used by Phillips Robbins, and he has found that this region of the column profile does not contain significant levels of a Ca2+-dependent GDPase activity, a probable Golgi marker enzyme (Phillips Robbins, personal communication), suggesting that our vesicle prepation from sec6-4 cells is free of Golgi contamination. The invertase activity present in the wild-type elution profile was much lower, due to the very rapid transit of invertase to the cell surface and the corresponding low abundance of secretory vesicles in wild-type cells. However, Figure 6B shows that the bulk of Sec4p present in the wildtype P3 pellet (10% of the total Sec4p) co-eluted with a small peak of invertase activity. The small apparent shift in the position of the Sec4p peak with respect to the invertase peak in Figure 6B is probably insignificant and attributable to error in the measurement of low levels of Sec4p and invertase activity. The invertase activity was

detected only in the presence of Triton X-100, indicating that the activity is intravesicular. No detectable plasma membrane marker activity was found in this region. The wild-type peak of invertase and Sec4p (Figure 6B) is four fractions later than the sec6-4 peak (Figure 6A), yet this is consistent with the observation that the vesicles that accumulate in sec6-4 cells are somewhat larger (100 nm) than the vesicles seen in wild-type cells (80 nm) (Walworth and Novick, 1987).

Localization of Sec4p by lmmunofluorescence We have used immunofluorescence as an independent means of localizing Sec4p. Wild-type and sec6-4 cells were fixed and processed for immunofluorescence after a 2 hr incubation at 37%. As a control, we have used purified IgG from a nonimmunized rabbit. The control IgG yields only faint diffuse staining of yeast cells (Figure 7A). Using the same concentration of affinlity-purified antiSec4p antibody, wild-type cells show several distinct dots per cell as well as a general stain that appears to be somewhat brighter than that of the control cells (Figures 7B-7D). The sec6-4 mutant cells exhibit a large number of dots per cell (Figures 7E-7H). These appear to fill the cell interior yet avoid the nuclear region. These observations are consistent with the hypothesis that the dots represent secretory vesicles. Taken together, the fractionation and immunofluorescence data suggest that Sec4p is associated with both the plasma membrane and secretory vesicles. In wild-type cells, the plasma membrane pool of Sec4p is predominant, but a minor pool is associated with another compartment, most likely secretory vesicles in transit to the plasma membrane. In sec6-4 cells, the Sec4p is depleted

Cell 758

Figure 6. Sec4p Co-Elutes with lnvertase Activity in P3 Pellets from se&-4 Cells (A) and Wild-Type Cells (E) Chromatographed on a Sephacryl S-1000 Column The P3 pellets were generated by differential centrifugation, resuspended in 1 ml of 0.9 M sorbitolll0 m M triethanolamine (TEA) (pH 7.5), and applied to a 1.5 cm x 90 cm Sephacryl S-1000 column. Material was eluted from the column at a flow rate of 9.2 mllhr and 4 ml fractions were collected. lnvertase and vanadatesensitive Mg s+ ATPase activities were measured and the amount of Sec4p was measured by quantitative Western blot. lnvertase (pmoles of glucose produced per min per fraction); vanadate-sensitive Mg *+ ATPase (nmoles of phosphate produced per min per fraction); Sec4p (cpm per fraction).

15

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Irxtrons

from the plasma membrane pool and it is mainly associated with the secretory vesicles that accumulate during the shift to 37°C. The vesicular pool can be cleanly separated from the plasma membrane-bound pool by differential centrifugation.

Sec4p Is Redistributed in sec6-4 Cells

onto Secretory Vesicles

We have examined the time dependence of Sec4p redistribution in sec6-4. Mutant cells were grown at 25% and then tranferred to 37% for various lengths of time. At each time point, an aliquot of cells was lysed and fractionated by differential centrifugation. As shown in Figure 8, the amount of Sec4p progressively increased in P3 pellets and concomitantly decreased in P2 pellets. As a control, we show that in wild-type cells, the distribution of Sec4p between P2 and P3 pellets did not change during the 2 hr shift to 37% One likely explanation of these data is that

Sec4p is depleted from the plasma membrane pool (P2 pellet) and redistributed onto the secretory vesicles (P3 pellet) as they accumulate in se&-4 cells at the restrictive temperature. In order to test this hypothesis, the following experiment was done. SeC6-4 cells were labeled with [35S]methionine overnight at 25% and then chased with excess cold methionine during a 2 hr shift to 37°C. The fraction of prelabeled Sec4p in the P3 fraction increased from 30% of Sl to 60% after the incubation at 37°C. These results suggest that a mechanism exists by which a major fraction of Sec4p can be retrieved from the plasma membrane and become associated with secretory vesicles. Such a mechanism could involve either membrane recycling or a soluble intermediate. Walworth and Novick (1987) have previously reported that, in a similar pulsechase experiment, the majority of vesicular proteins are synthesized as the vesicles are formed. This result excludes extensive recycling of plasma membrane proteins

Localization 759

of Sec4 Protein

Figure 7. lmmunofluorescence

Localization

of Sec4p

Wild-type (A-D) or sec6-4 cells (E-H) were grown at 25’C in medium containing 2% glucose then shifted to medrum containing 0.2% glucose and incubated at 37% for 2 hr. The cells were fixed and processed for immunofluorescence. Cells were stained with 20 Kg/ml of control IgG (A) or affinitypurified antiSec4p antibody (S-H)

into vesicles during the shift to 37%. We therefore favor the hypothesis that Sec4p is dissociated from the plasma membrane and redistributed onto the vesicles through a soluble intermediate. We have assessed the location of Sec4p in the eight other secretory mutants that accumulate vesicles at their

40 G % R

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IO

1‘20

Time at 37OC ( min > Figure 8. Sec4p Is Redistributed

onto Secretory Vesicles

Sec6-4 cells were grown at 25% in YP 2% glucose and then transferred to 37% for various lengths of time in YP 0.2% glucose. At each time point, aliquots of cells were lysed osmotically and the lysates were fractionated by differential centrifugation. Samples of P2 and P3 pellets were run on 10% SDS gels and transferred onto nitrocellulose. The amount of Sec4p was quantitated using ‘ssl-Protein A. The results are expressed as the percentage of Sec4p (cpm) found in Sl supernatants. The amount of Sec4p progressively increases in P3 pellets (0) and concomitantly decreases in P2 pellets (+). In wild-type cells, the distribution of Sec4p within P2 (0) and P3 (0) pellets does not change before and after a 2 hr shift at 37%.

restrictive temperature (Novick et al., 1980). After transfer for 2 hr to 37% in 0.2% glucose, the cells were lysed and the lysates were fractionated by differential centrifugation as described in Figure 2. Table 2 shows that the distribution of Sec4p between P2 and P3 pellets was similar in these eight strains to that seen in sec6-4 cells (Figure 3), suggesting that in these cells as well, thle majority of the Sec4p is associated with the accumulating vesicles. Control experiments have been performed with sec7-7 and sec78-7 cells that respectively develop Golgi-like and ERlike organelles when transferred to the restrictive temperature (Novick et al., 1980). These organelles contain invertase as a result of the transport block. We have fractionated the P2 pellets from these strains on sucrose gradients. In both

Table I. Strains Used Strain

Genotype

NY3 NY13 NY17 NY57 NY61 NY64 NY130 NY176 NY399 NY405 NY410 NY412 NY432 NY451 NY456 NY494 NY499

MATa, MA Ta, MA Ta, MATa, MATa, MATa, MATa, MATa, MATu, MA Ta, MATa, MATa, MATa, MATa, MATa, MATa, MATa,

ura3-52, secl-1 ura3-52 ura3-52, sec6-4 ura3-52, sec9-4 ura3-52, seclO-2 ura3-52, set 15-1 ura3-52, sec2-41 ura3-52, sec7-1 ura3-52, sec5-24 ura3-52, sec4-8 ura3-52, sec8-9 ura3-52, sec3-2 ura3-52, secl8-1 ura3-52, Gal+ ura3-52, Gal+, sec4-8 ura3-52, Gal’, SEC4::(URA3, GAL 1.SEC4) ura3-52, Galf, sec4-8::(URA3, GAL 1-seed-8)

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Table 2. Distribution

should be sensitive to protease digestion in the absence as well as in the presence of detergent. Figure 9 shows the result of such an experiment. After treatment of vesicles with either trypsin or proteinase K, Sec4p was degraded to the same extent regardless of the presence of Triton X-100, indicating that Sec4p is exposed on the vesicle surface. Under these conditions, protease treatment does not affect the integrity of the vesicles (Walworth and Novick, 1987). To determine the orientation of Sec4p with respect to the plasma membrane, we have performed protease protection studies using intact spheroplasts. Wild-type cells were converted to spheroplasts and treated with proteinase K. Under these conditions, the Sec4p was not degraded, indicating that it is located on the cytoplasmic face of the plasma membrane (A. Kabcenell, unpublished data).

of Sec4p in Various Strains ;21,

s3 (%Sl)

P3 (%Sl)

32

51

15

12

secl-1 sec2-4 sec3-2 sec5-24 sec8-9 sec9-4 seclO-2 sec751

70 64 59 57 52 62 56 61

23 27 21 21 25 19 22 30

16 17 14 15 11 13 17 20

46 49 45 51 54 49 46 44

sec7-1 secl8-1

20 34

54 49

10 ND

4 ND

Strain

s2 (%Si)

SEC+

Cells were grown at 25°C in YP 2% glucose and transferred to 37’% for 2 hr in YP 0.2% glucose. Ceils were lysed osmotically and the lysates were fractionated by differential centrifugation. Aliquots of the different supernatants and pellets were run on 10% SDS-PAGE gels and transferred onto nitrocallulose. Sec4p was quantitated in each fraction using ‘ssl-Protein A. The results are expressed as the percentage of Sec4p found in Sl. ND= not done.

Sec4p Behaves as an Integral Membrane Protein To investigate the nature of the interaction between the Sec4p and the plasma membrane or the secretory vesicles, a variety of extraction procedures have been tested. We have used in these experiments either a P2 pellet from wild-type cells or purified secretory vesicles from sec6-4 cells. The same pattern of solubilization was obtained with either subcellular fraction. Table 3 shows the results obtained with the secretory vesicles. Samples were treated with various reagents, spun at 100,000 x g and the Sec4p in the supernatants and pellets was quantitated by Western blot analysis. Sec4p was readily extractable by lo/o Triton X-100, but was not extracted by either 1 M NaCl or 6 M urea. Treatment at high pH resulted in a loss of material, most likely due to proteolytic degradation, but the remaining Sec4p was mostly found in the pellet. We have also treated the secretory vesicles with 1 M hydroxylamine, (pH 8.0) which cleaves the thioester bonds between fatty acids and proteins (Magee and Courtneige, 1985). As observed with high pH treatment, about 50% of total Sec4p was lost.

cases, we found that most of the invertase activity and Sec4p was present in the P2 pellet (Table 2). However, Sec4p did not co-migrate with invertase activity on the density gradients, but remained predominantly associated with the plasma membrane, as in wild-type cells (data not shown). Thus, the shift in Sec4p distribution from the plasma membrane is specific to vesicle accumulating set mutants. Sec4p Is Present on the Cytoplasmic Face of Secretory Vesicles To address the topology of Sec4p membrane association, protease protection experiments were performed on secretory vesicles purified from sec6-4 cells on an S-1000 column. If the Sec4p is on the outside of the vesicles, it

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Figure 9. Sec4p Is Present on the Cytoplasmic

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+

Face of Secretory Vesicles

The fractions 21-24, generated by chromatography of P3 pellet from se&4 cells on the Sephacryl S-1000 column (see Figure 6) were pooled, centrifuged for 1 hr at 100,000 x g. and resuspended in 1 ml of 0.8 M sorbitol/TEA. Aliquots were incubated on ice with O-50 Kg/ml of trypsin (right panel) or O-5 Kg/ml proteinase K (left panel). Samples incubated with trypsin or proteinase K in the presence of detergent were first preincubated with 0.05% Triton X-100. As a control, some samples were incubated in the presence of 100 uglml of trypsin inhibitor or PMSF (10e3 M). After 1 hr of incubation, samples were boiled in SDS and run on a 10% SDS gel. Sec4p was revealed after transfer onto nitrocellulose using the anti-Sec4p antiserum (dilution l/1000) and a goat anti-rabbit IgG alkaline phosphatase conjugate. The Sec4p is equally sensitive to proteases regardless of the presence of detergent, indicating that Sec4p is on the outer surface of secretory vesicles.

iocaiization 761

of Sec4 Protein

Table 3. Extraction of Sec4p from Secretory Vesicles

control NaCl (1 urea (6 TX 100 NHPOH Na2C03

Pellet

Supernatant

Treatment ~~~--

M ) M) (1%) (1 M) (0.2 M) (pH 11 .O)

Recovery

-- _--

cm

%

cm

%

w

%

660 650 658 2982 555 72

18 21 19 88 36 5

2925 2378 2820 421 985 1424

a2 79 ai 12 64 95

3595 3028 3478 3403 1540 1496

100 a4 97 95 43 42

The fractions 21-24 generated by chromatography of P3 pellet from sec6-4 cells on the Sephacryl S-1000 column (see Figure 6) were pooled, divided into six aliquots, and ultracentrifuged for 1 hr at 100,000 x g. Each pellet was resuspended in 0.8 M sorbitol/TEA (control), 1 M NaCI, 1% Triton X-100 or 6 M urea made in 0.8 M sorbitol/TEA, and 0.2 M sodium carbonate (pH 11 .O) (in water) or 1 M hydroxylamine (in water). After 15 min on ice or 1 hr at 37°C (hydroxylamine), the samples were spun for 1 hr at 100,000 x g. The supernatants and pellets were boiled in SDS and run on a 10% SDS-PAGE gel, After transfer to nitrocellulose, Sec4p was quantitated using 1*51-Protein A. The results are expressed as total cpm found in each fraction.

Of the remaining Sec4p, two-thirds was associated with the insoluble fraction. The above data suggest that the Sec4p is not loosely attached to the membrane, but rather that it exhibits the solubility properties of an integral membrane protein. The behavior of Sec4p following extraction with Triton X-114 has also been monitored. This detergent forms an organic phase at 30°C, and most hydrophobic proteins partition into this detergent phase, while most hydrophilic proteins partition into the aqueous phase (Bordier, 1981). In ihe experiment presented in Table 4, membrane-bound Sec4p from wild-type cells partitioned into the detergent phase, whereas the small amount of Sec4p present in the high speed supernatant partitioned into the aqueous phase. A similar experiment was performed with an overproducing strain (NY494). When Sec4p was overproduced by 11-fold, 60% of the Sec4p was found in the high speed supernatant. As shown in Table 4, this pool partitioned into the aqueous phase, while most of the membrane-bound form partitioned into the detergent phase. These results indicate that the pool of Sec4p found in high speed supernatants behaves as a hydrophilic protein, while the Sec4p in the high speed pellets behaves as a hydrophobic protein.

No difference in electrophoretic mobility on 15% SDS gels has been detected in these experiments between the soluble and membrane-bound Sec4p (data not shown). The Sec4p does not possess an apparent signal sequence and encodes a generally hydrophilic protein (Salminen and Novick, 1987). This protein must therefore undergo some posttranslational modification(s) or conformational change in order to be attached to the plasma membrane or to the membrane of the secretory vesicles. It has been suggested that fatty acid acylation of some proteins including the ras gene produlcts may mediate their membrane attachment (Magee and Schlesinger, 1982; Sefton et al., 1982). In the case of ras proteins, a palmitate moiety is added in a thioester linkage to a cysteine located four residues from the carboxy end of the protein (Willumsen et al., 1984b; Chen et al., 1985; Buss and Sefton, 1986). In contrast, Sec4p terminates in two cysteines. It is possible that one of these residues is modified by the addition of palmitic acid. We have tested this possibility by labeling wild-type cells and cells that overproduce Sec4p with [3H]palmitate. The lysates were then immunoprecipitated using the anti-Sec4p antibody. However, despite various attempts, we have not been able to

Table 4. Behavior of Sec4p Following TX 114 Extraction Wild Type (34772 cpm per 1 OD U) Supernatant tw

Pellet to4

+

Supernatant tom

a2

12 Aqueous tow

Sec4p Overproducer (380768 cpm per 1 OD U)

Detergent W)

Pellet w

58

39

Aqueous PM

Detergent PJ)

Aqueous to/a)

Detergent tw

Aqueous to4

Detergent (Q/a)

16

61

106

6

35

61

Wild-type cells and cells expressing normal Sec4p behind the GAL7 promoter were osmotically lysed in 0.8 M sorbitol/TEA. Cell debris was removed at 450 x g, and the low speed supernatant (Sl) was spun at 100,000 x g. The high speed supernatant and pellet (resuspended in 0.8 M sorbitol/TEA) were treated for 2 min at 4’YI and for 1 min at 30°C with an equal volume of 2% Triton X-l 14 (in 0.3 M NaCl and 0.8 M sorbitol/TEA). Samples were then spun for 1 min in a microfuge to separate the detergent and aqueous phases. After boiling in SDS, aliquots of the different samples were run on a 10% SDS-PAGE gel, transferred onto nitrocellulose, and the amount of Sec4p was quantitated. The results are expressed as the percentage of Sec4p found in Sl, except for the supernatant of wild-type cells in which the Sec4p was not sufficiently abundant to be quantitated.

Ceil 762

Table 5. Newly Synthesized

Sec4p

IS

Rapidly Attached to Both the Plasma Membrane 1 Min Pulse

and the Secretory Vesicles 1 Min Pulse Plus 1 Hr Chase

Genotype

Sec4p Fraction

P2 (%Sl)

;27,

P3 (%Sl)

P2 (%Si)

;iSl)

SEC+

newly synthesized

48

35

37

a2

20

17

W) total (lz51-Protein A)

52

12

11

59

15

ia

newly synthesized

26

29

31

37

15

55

PS) total (‘*sl-Protein

30

11

45

15

13

52

se&-4

A)

Wild-type and sec6-4 cells were labeled for 1 min with 1 mCi of [ssS]methionine (as described in Experimental Procedures) and chased for 1 hr with an excess amount of cold methionine. The cells were converted to spheroplasts, lysed, and the lysates were fractionated by differential centrifugation. Aliquots of the different supernatants and pellets were immunoprecipitated with antiSec4p antibody. The amount of radiolabeled Sec4p in each fraction has been estimated by gel densitometry. To calculate the total amount of Sec4p (steady state), aliquots of the different samples were displayed by SDS-PAGE and transferred onto nitrocellulose. Sec4p was quantitated using ‘ssl-Protein A. The results are expressed as the percentage of Sec4p found in Sl supernatants.

demonstrate that the Sec4p can be labeled with 13H]palmitate (data not shown). The RAM7 gene (also called DHRI) has been implicated in the membrane attachment of the RAS2 gene product, in the export of the mating pheromone a factor, and in the palmitylation of a set of yeast proteins (Powers et al., 1986; Fujiyama et al., 1987). We have tested the effect of the ram&l temperature-sensitive mutation on Sec4p membrane attachment and electrophoretic mobility. No change in either of these parameters was observed following incubation at the restrictive temperature, indicating that the localization of Sec4p is not dependent on the RAM7 gene product (data not shown).

Sec4p Rapidly Associates with Membranes We have probed the biosynthetic pathway of Sec4p by pulse-chase experiments. Wild-type and sec6-4 cells were shifted to 37°C for 30 min and pulse-labeled for 1 min with [35S]methionine. The ceils were then chased for 1 hr at 37% with an excess of cold methionine, washed at 4°C in the presence of sodium azide and cycloheximide, converted to spheroplasts, and lysed. The lysates were fractionated as described above, and aliquots of the different supernatants and pellets were immunoprecipitated using the anti-Sec4p antibody, and the precipitates were displayed by SDS-PAGE. The amount of newly synthesized Sec4p in each fraction was determined by densitometry of the resulting autoradiogram, while the total amount of Sec4p was quantitated by Western blot analysis using lz51-Protein A. The results are summarized in Table 5. After a 1 min pulse, newly synthesized Sec4p was detected in P2, S3, and P3 from wild-type cells. Sec4p was relatively more abundant in 53 and in P3 after a 1 min pulse than it was at steady state (total Sec4p). Following the chase, the P2 pool increased and only a small amount of SecCp was detectable in S3 and P3. In sec6-4 cells at the restrictive temperature, newly synthesized Sec4p was found in roughly equal amounts in P2, S3, and P3 after a 1 min pulse. After 30 min at 37°C

most of the total Sec4p (as revealed by immunoblot) was equally distributed between the P2 and P3 pellets, which is consistent with the data presented in Figure 8. During the chase, the pool of pulse-labeled Sec4p increased in the P3 pellet as did the pool of total Sec4p. The amount of pulse-labeled Sec4p present in S3 diminished during the chase, as observed in wild-type cells. These results are consistent with the hypothesis that at least a part of Sec4p present in S3 represents a soluble precursor form of Sec4p. However, the membrane attachment process is very fast. Newly synthesized Sec4p can probably attach to either the plasma membrane (P2) or the secretory vesicles (P3). To rule out the possibility that Sec4p redistributed during spheroplast formation, cells were disrupted with glass beads immediately after pulse or chase. Crude membrane and soluble fractions were generated by high speed centrifugation, essentially as described by Fujiyama and Tamanoi (1986). As shown in Figure 10, the majority of the Sec4p was found in the membrane fraction even after only a 1 min pulse and rapid mechanical breakage. The soluble pool of Sec4p greatly decreased after a 1 hr chase, suggesting that it represents the precursor form of membrane-bound Sec4p. The same results were obtained after a 5 min pulse and a 1 hr chase. These data confirm the observation that newly synthesized Sec4p very rapidly associates with membranes (tIj2 < 1 min). Furthermore, it should be noted that no shift in mobility was observed between newly synthesized soluble and membrane-bound forms of Sec4p on a 15% SDS gel (Figure 10). The very rapid membrane association of Sec4p would, in the absence of any opposing dissociation reaction, lead to the disappearance of the soluble pool. Nonetheless, 15% of Sec4p formed a truly soluble pool at steady state or following a 1 hr chase (Table 5). These results suggest that a dissociation reaction may exist. Such a dissociation reaction may play a role in the redistribution of Sec4p seen during vesicle accumulation. However, we cannot exclude the possibility that dissociation occurs only during lysis or fractionation.

Localization 763

of 9x4

Protein

L

s

P

L

s

P

L

s

Figure 10. Newly Synthesized Sec4p Rapidly Becomes Membrane-Bound

5 min pulse + lh chase

5 min pulse

1 min pulse + lh chase

1 min pulse

P

Discussion We have previously cloned and sequenced the SfC4 gene, one of the 10 genes involved in the secretory pathway between the Golgi apparatus and the plasma membrane in Saccharomyces cerevisiae (Salminen and Novick, 1987). In the present report, we have characterized Sec4p with regard to its intracellular location and biochemical properties. These data shed new light on the celMar function of this protein. SEC4 encodes a protein that belongs to the superfamily of ras proteins. These proteins have in common intrinsic biochemical properties that are similar to those of regulatory G proteins involved in signal transduction across the cellular membrane (Gilman, 1987). The most distinctive property is the ability to bind and hydrolyze GTP in conjunction with the transduction of a regulatory signal. Sec4p displays a high degree of sequence similarity to the family of ras proteins in the four main regions thought to collectively participate in guanine nucleotide binding activity and GTPase activity, which are required for ras function (Barbacid, 1987). We directly demonstrate here that the Sec4p is a GTP-binding protein (Figure 1). It is likely that Sec4p also displays a GTPase activity, and we are currently testing this hypothesis. We have found in this study that the mutant Sec4p encoded in the sec4-8 strain does not bind detectable levels of GTP, even when overproduced behind the strong GAL1 promoter (Figure 1). It should be noted that the mutation in this protein is not located in either of the two regions thought to participate in the binding of the guanine ring (Walter et al., 1986; Sigal et al., 1986). It is tempting to speculate that the secretory defect of sec4-8 cells is directly related to an in vivo impairment of GTP-binding by Sec4p. However, since we have measured the ability of Sec4p to bind GTP after transfer to nitrocellulose filters, we cannot rule out the possibility that it is the renaturation behavior on filters rather than GTP-binding that distinguishes the mutant protein from normal Sec4p. Furthermore, the phenotype of sec4-8 may be, at least in part, a consequence of the lowered levels of Sec4p present in this strain, a situation resulting from

L

S

P

Very

Wild-type yeast were labeled for 1 minor 5 min with 1 mCi of [%]methionine, and aliquots were chased for 1 hr as described in Experimental Procedures. Cells were then broken with glass beads and centrifuged for 10 min at 1500 x g to remove cell debris. The lysates (L) were spun for 1 hr at lOO~,OOO x q to generate crude soluble (S) and membrane (P) fractions. Equal aliquots of the different fractions were then immunoprecipltated with the anti-Sec4p antibody and run on a 15% SDS gel. After a 1 min pulse, the majority of Sec4p is already associated with the membrane fraction. Note also that the soluble pool of SecJp displays the same apparent electrophoretic mobility as membrane-bound Sec4p and that this soluble pool decreases after 1 hr of chase.

the instability of the altered protein. Supporting this possibility, we have found that overproduction of the mutant protein restores growth at the restrictive temperature (A. Salminen, unpublished data). The intracellular localization of Sec4p difiers from that of other ras proteins studied to date. Most ras proteins have been localized to the inner face of the plasma membrane (Willingham et al., 1980; Willingham et al., 1984; Fujiyama and Tamanoi, 1986). A major conclusion of this study is that Sec4p is associated not only with the plasma membrane but also with secretory vesicles. This association with vesicles becomes particularly dramatic in set mutant cells that accumulate intracellular vesicles following ashift to the restrictive temperature. The accumulation of secretory vesicles in the mutant cells and their uniform size allow for very effective purification from other membranes (Figure 6A; Walworth and Novick, ‘1987; Phillips Robbins, personal communication). Therefore, we can conclude with high confidence that Sec4p is associated with the accumulated vesicles. The punctate staining pattern seen by immunofluorescence supports this conclusion (Figures 7E-7H). Even in wild-type cells, we have been able to show that a pool of Sec4p is probably associated with the small population of secretory vesicles in rapid transit to the plasma membrane. These wild-type vesicles are the same density yet are slightly smaller in size than the accumulated vesicles in the mutant cells (Figures 5 and 6). Association of Sec4pl with the plasma membrane is expected since the membrane of the secretory vesicles represents the precursor to this organelle. While purification of the plasma membrane is somewhat more problematic, we have achieved IO-fold purification of the plasma membrane and have observed excellent cofractionation of Sec4p with the plasma membrane marker enzyme, vanadate-sensitive ATPase. The plasma membrane fraction is at least partially resolved from the other major organelles of the cell. Also, exaggeration of either the ER or Golgi in an appropriate mutant strain does not result in a shift in distribution from the plasma membrane. While most of the Sec4p appears to be tightly bound to the plasma membrane or secretory vesicles and behaves

Cell 764

as an integral membrane protein (Tables 2 and 3), the Small amount of Sec4p found in high speed supernatants acts as a truly soluble protein, and it partitions into the aqueous phase following solubilization with Triton X-114 (Table 4). Since the protein sequence of Sec4p is hydrophilic (Saiminen and Novick, 1987), a portion of this pool probably represents the precursor form of Sec4p prior to association with membrane compartments. This hypothesis is supported by the fact that the newly synthesized soluble pool is relatively more abundant than the soluble pool found at steady state, and it decreases during a chase experiment (Table 5 and Figure 10). It must be noted that no difference in electrophoretic mobility was observed between soluble and membrane-bound forms of Sec4p (Figure 10). This contrasts with other ras proteins for which the membrane-bound and processed forms exhibit a faster migration rate than their unmodified cytosolic counterparts (Shih et al., 1982; Buss and Sefton, 1986; Fujiyama and Tamanoi, 1986). Newly synthesized Sec4p appears to be very rapidly associated with membranes, as shown in a 1 min pulse. This process is more rapid than the attachment of the yeast RASl and RAS2 gene products In those cases, only soluble forms are detected after a 1 min pulse (Fujiyama and Tamanoi, 1986). The attachment of mammalian ras proteins to the plasma membrane also appears to be a much slower process (Shih et al., 1982). Newly synthesized Sec4p is probably inserted simultaneously onto both the plasma membrane and secretory vesicles. Thus, some of the plasma membranebound Sec4p is directly attached from the soluble pool, while another fraction may be brought by exocytic fusion of secretory vesicles. How does the Sec4p become membrane-bound? We have shown that membrane-bound Sec4p partitions into TX-114, which clearly indicates that the Sec4p must undergo either a covalent modification or a conformational change in order to be inserted into membranes. The fact that the soluble pool of Sec4p increases dramatically when Sec4p is overproduced suggests that membrane attachment is a saturable process (Table 4). Mammalian Ras proteins as well as yeast Raslp and RasPp are modified by the covalent addition of a palmitate moiety that appears to mediate their insertion into membranes (Chen et al., 1985; Buss and Sefton, 1986; Fujiyama and Tamanoi, 1986). However, we have failed to detect any palmitylation of the Sec4p (data not shown). We have also found that Sec4p remains membrane-bound in cells that carry a mutation in the RAM7 gene, which is required for the acylation and membrane localization of Raslp and RasPp (Powers et al., 1986; Fujiyama et al., 1987; data not shown). Furthermore, the acylation of Ras proteins may cause a shift in electrophoretic mobility between soluble and membrane forms (Willumsen et al., 1984b; Deschenes and Broach, 1987), which is not observed in the case of Sec4p. The possibility remains that Sec4p is too minor a protein for palmitylation to be detected or that the fraction of the population of Sec4p containing palmitic acid is low, as has been shown for ~21 ras (Buss and Sefton, 1986). However, Sec4p does not possess the consensus sequence for palmitylation found in other ras proteins: Cys-A-A-X, where

A is any aliphatic amino acid and X is often the carboxy terminal residue. This cysteine residue is the site of palmitylation (Willumsen et al., 1984b; Chen et al., 1985), and mutations that replace this cysteine render the protein unable to bind tightly to membranes and reverse its transformation potential (Willumsen et al., 1984a), but the role of the two aliphatic amino acids is not known. Whether the two cysteines found at the carboxy end of the Sec4p (Salminen and Novick, 1987) are involved in membrane attachment will be addressed in future experiments by constructing mutant molecules lacking these two residues, Our previous study on the SEC4 gene established that it plays an essential role in the control of a late stage of the secretory pathway (Salminen and Novick, 1987). It is generally though that ras proteins, by analogy with G proteins, participate in the transduction of an external signal across the cellular membrane (Gilman, 1987). Such a role has been clearly demonstrated for yeast Raslp and Ras2p, which activate adenylate cyclase in response to nutrient signals (Toda et al., 1985). Such a function is consistent with the localization of ras proteins at the inner face of the plasma membrane. Because secretion in yeast appears to be constitutive and does not require any known external stimulus, there is no obvious need for such a transmembrane signal transduction system. Since secretion is spatially controlled in yeast (Field and Schekman, 1980), one possible role for the plasma membrane-bound pool of Sec4p is to confine the site of vesicle fusion to the bud. However, the finding that a portion of the Sec4p is on secretory vesicles suggests another function for this GTP-binding protein; Sec4p could in fact function on the vesicle surface to transduce an intracellular signal needed to regulate the traffic of vesicles between the Golgi apparatus and the plasma membrane. The presence of Sec4p on the secretory vesicles may allow it to directly or indirectly control the movement of secretory vesicles and/or regulate the fusion of the vesicles with the plasma membrane. However, we do not exclude the possibility that Sec4p transiently associates with the Golgi apparatus or a specialized subcompartment of the Golgi during its function. Recent data from a number of laboratories suggest that additional GTP-binding proteins may act either at various levels of the constitutive secretory pathway or to control regulated secretion in some specialized cells. Using an in vitro assay, Melancon et al. (1987) showed that nonhydrolyzable GTP analogs inhibit the transport between Golgi subcompartments and thereby cause the build-up of vesicles. In this case, the hypothetical G protein has been proposed to function on the acceptor membrane in an inhibitory fashion. Segev et al. (1988) constructed a cold-sensitive mutation in the yeast YfT7 gene and found that it causes accumulation of incompletely glycosylated invertase and a build-up of the ER membranes at the restrictive temperature, suggesting a role for this Sec4prelated G protein in vesicular transport to the Golgi apparatus. In permeabilized neutrophils or RlNmF cells, GTP analogs can stimulate the secretory process in a calcium-independent fashion (Barrowman et al., 1986; Vallar et al., 1987). In mast cells, a similar effect is ob-

Locallzatlon 765

of Sec4 Protein

served but in synergy with calcium (Howell et al., 1987). These observations led Gomperts and co-workers to postulate the existence of a novel G protein, Ge, that would directly activate the exocytic mechanism. Interestingly, they have hypothesized that this G protein may be on the surface of secretory granules (Gomperts, 1986). It will be of interest to identify and localize these GTPbinding proteins. One can speculate that they too will be found to be uniquely localized to a particular compartment of the secretory pathway. Such a finding would support a role for this family of G proteins in maintaining the orderly flow of vesicular traffic. Experimental Procedures Yeast Strains and Growth Conditions Yeast strains used in this study are listed In Table 1. Cells were grown in rich medium (YPD) containing 1% Bacto Yeast extract, 2% Bacto Peptone (Difco), and 2% glucose, or in minimal medium (SD) containing 0.7% Yeast Nitrogen Base without amino acids (Difco), 2% glucose, and supplemented for auxotrophic requirements as described by Sherman et al. (1974) when necessary. Yeast transformation was done by the method of alkali cation treatment (Ito et al., 1983). Transformants were selected on SD medium at 25°C. Nucleic Acid Techniques Bacteria and plasmid constructions were done as described earlier (Salminen and Novick, 1987). Plasmid pNB139 contains the SEC4 gene on a 1.4 kb EcoRI-BamHI fragment. Plasmid pATH2 is an inducible expression vector containing the truncated TrpE gene (1 Koerner and A. Tzagaloff, personal communication). Plasmid pNB153, which contains the TrpE-SEC4 fusion, was constructed as follows: pATH2 was digested with restriction enzymes Smal and Pvul to open the vector, and pNB139 was digested with Pvul and EcoRV (Pvul cuts in the B/a gene in both plasmids). Fragments were electrophoresed in a 0.8% agarose gel and purified from agarose by freezing and thawing in phenol (Benson, 1984). The purified fragments were religated and used to transform E. coli (DHI). Transformants were plated on LB plates containing ampicillin (100 pg/ml) to select for reconstruction of the B/a gene and tryptophan (40 pglml) to repress synthesis of the fusion protein. Plasmid pNB170 was constructed by digesting pNB139 with BamHl and Pvull. The isolated fragment was further digested with SaulllA. The new SaulllA-Pvull fragment was religated with the vector portion of the above BamHI-Pvull digestion. This removes 250 bp from the leader sequence of the SEC4 gene, and fortuitously recreates the BamHl site. Plasmid pNB 187 was constructed by inserting an 820 bp EcoRI-BamHI fragment containing the control region of the yeast GAL7 and GAL70 genes (Johnston and Davis, 1984) into Ycp50, a yeast centromere plasmid constructed by C. Mann; for map see Kuo and Cambell (1983). Plasmid pNB187 allows expression under GAL7 control of genes inserted into the BamHl site. To clone SEC4 under the GAL7 control, plasmid pNB170 was cut with BamHl and subjected to Bal31 digestion. The digests were pooled and religated in the presence of a synthetic linker (dpGGAATTCC; New England Biolabs #1020) and used to transform E. coli. A number of trahsformants were screened for plasmids with proper insert size by EcoRI, and EcoRI-Hindlll digestions. Based on this rough estimate of insert size, several plasmids were digested with EcoRI-Hindlll, and the isolated fragments, containing the amino end of the gene, were cloned in Ml3 phage derivative mp19 for sequencing (see Salminen and Novick, 1987). A clone was found that contained eight nucleotides upstream from the ATG codon of SEC4. This plasmid was designated pNB188. To construct plasmid pNB190, which contains the SEC4 gene under GAL7 control, pNB187 was digested with BamHl and pNB188 with EcoRI. The recessed ends of vector and insert were filled in by Klenow fragment (Boehringer Mannheim), and both were electrophoresed in agarose gel. After purification from an agarose gel, the ends of the vector were dephosphorylated with calf intestinal phosphatase (Boehringer Mannheim) and then blunt-end ligated with the insert. The GALI-SEC4 joint was verified by sequencing. Plasmid pNB191 was constructed from Ylp5

(Struhl et al., 1979) by removing the Pvull site by brief Bal31 digestion. integrating plasmid pNB219, containing SEC4 under GAL7 control, was constructed by cloning the 2.1 kb EcoRI-Sail fragment from pNB190 In EcoRI-Sail sites of pNB191. Preparation of the TrpE-Sec4 Fusion Protein and Rabbit Immunization The fusion protein was produced in E. coli strain, NRB153 (a DHl transformant containing the plasmid pNB153) grown in M9 CA medium containing ampicillin (100 pglml) and tryptophan (40 Nglml) at 37°C. An overnlght culture was diluted 1:lO in 10 ml of the same medium and grown to ODeoOof 1.0. The cells were placed in 200 ml of the medium wlthout tryptophan and grown for 75 min. lndoleacrylic acid (Sigma; 5 mglml in ethanol) was added to a final concentratior! of 20 pg/ml, and ceils were grown for 4 hr. The fusion protein was isolated from the insoluble fraction essentially as previously described (Kleid et al., 1981; Spindler et al., 1984). The fusion protein was electroeluted at 100 V from gel cubes in dialysis buffer and collected on dialysis membrane. The fusion protein in dialysis buffer was stored at -20°C (Hunkapiller et al., 1983). For immunization, 100 ~1 of the preparation (15 wg) was diluted to 0.5 ml with PBS and emulsified with 0.5 ml of Freund complete adjuvant for the initial injections into the popliteal lymph nodes of anesthetized rabbits. The immunization scheme was essentially as described by Louvard et al. (1982). Expressing SEC4 from the GAL7 Promoter To construct strains expressing SEC4 from the GAL7 promoter, a GAL+ yeast strain NY451 was transformed with plasmid pNB219. The plasmid was linearized prior to the transformation by digesting with Pvull. Upon transformation, the plasmid integrates into the genome by homologous recombination creating a duplication of the chromosomal copy (Orr-Weaver et al., 1981; Salminen and Novick, 1987). In the case of plasmid pNB219, the second copy of the gene is under GAL7 control (data not shown). When cells grow in medium containing glucose, the GAL promoter remains repressed (Adams, 1972), and only the copy under the normal SEC4 is expressed. The GAL7 controlled expression can be induced upon growth on galactose to overexpress Sec4p. To overexpress the mutant sec4-8 gene product, a Gal+ sec48 strain, NY456 was transformed with plasmid pNB219. In this case the plasmid was linearized by digesting with Hindlll-Pvull (this region contains the sec4-8 mutation; Salminen and Novick, 1987). When this gapped-linear plasmid integrates into the chromosome, the missing segment is repaired from the chromosomal information (Orr-Weaver et al., 1981), creating a similar situation as above: allowing the mutant sec4-8 gene product to be overexpressed upon growth on galactose. To induce the GAL7 promoter, cells were grown on YP medium, containing 2.5% ethanol, 2.5% glycerol, and 2% lactate to an ODsoo of 0.5, after which galactose was added directly to the medium to a concentration of 2%. Cell Fractionation Cells (usually 100-200 OD,,, U) were grown ar 25°C in YP medium containing 2% glucose. In most of the experiments, cells were pelleted and transferred for 2 hr to 37% in YP supplemented with 0.2% glucose. After washing in cold 10 m M azide, cells were resuspended in 6-12 ml of spheroplast medium (1.4 M sorbitol, 50 m M KPi [pH 7.51, 10 m M azide, 40 m M P-mercapthoethanol, and 1.5-3.0 mg of zymolyase-100T [Seikagaku Kogyo Co; ICN]) and converted to spheroplasts during a 45 min incubation at 37%. The spheroplasts ‘were resuspended in 1.5-3.0 ml of lysis buffer (0.8 M sorbitol in 10 m M triethanolamine, 1 m M EDTA [pH 7.21) containing phenylmethyl sulfonyl fluoride, leupeptin, chymostatin, pepstatin, and antiparin, homogenized 20 times using a 2 ml Wheaton tissue grinder, and centrifuged at 450 x g for 3 min. The pellet (PI) was washed once in the same volume of lysis buffer and the supernatants were pooled (Sl). The Sl supernatant was spun at 10,000 x gav in an SW50.1 rotor (Beckman) for 10 min at 4°C. The pellet (PZ) was resuspended in lysis buffer. The 52 supernatant was further centrifuged at 100,000 x gav in the same rotor for 1 hr at 4°C and the pellet was resuspended in lysis buffer. In some experiments, the P2 and P3 pellets were resuspended in 60% sucrose (wt/wt) and subfractionated on 35%-60% sucrose gradients essentially as according to Ruohola and Ferro-Novick (1987). The fractionation of the P3 pellets

Ceil 766

on a Sephacryi S-1000 column has been performed scribed (Walworth and Novick, 1987).

as previously de-

lmmunofluorescence Wild-type and sec6-4 cells were grown at 25°C in YP medium containing 2% glucose and then transferred to YP medium containing 0.2% glucose and incubated for 2 hr at 37°C. After washing, cells were fixed for 90 min at room temperature with 3% paraformaldehyde and processed for immunofluorescence as previously described (Novick and Botstein, 1985). We have used in this experiment affinity-purified antiSec4p antibodies. For affinity purification, TrpE-Sec4 fusion protein was covalently linked to glutaraldehyde-activated polyacrylamide agarose beads (ACA22;LKB), and the antibodies were eluted from the immunoadsorbent according to Guesdon and Avrameas (1976). Electrophoresis, Immunoblotting, and GTP-Binding For SDS-polyacrylamide gel electrophoresis, samples were heated for 3-5 min at 100°C in sample buffer containing 2% SDS and run on 10% or 15% slab gels according to Laemmli (1970). After transfer onto nitrocellulose (BA 83,0.2 Km; Schleicher and Schuell) overnight at 4’%, Sec4p was probed with antiserum and radioiodinated staphylococcal protein A (30 mCi/mg; Amersham) by the following method adapted from Burnette (1981). Briefly, the blotted filters were incubated for 30 min in TBS (10 m M Tris, 150 m M NaCl lpH7.51) containing 1% gelatin and 0.5% bovine serum albumin (BSA) and for 30 min in the same buffer supplemented with 0.1% Tween 20. The rabbit antiserum against Sec4p (dilution 1:400 in TBS-gelatin, BSA-Tween) was added for 90 min. After washings in TBS-Tween, ‘a51-Protein A (0.5 &i/ml in the same buffer as for primary antibody) was added for 2 hr. Following washings, the filters were dried and autoradiographed for 1-24 hr. To quantitate the amount of Sec4p, pieces of nitrocellulose corresponding to the Sec4p band were cut out and counted. The linear range of this assay has been monitored using wild-type lysates and was found to cover the range from 0.5-20.0 Kg of total protein This range includes all of the data points in the experiments reported. In some experiments, Sec4p was probed with the antiSec4p antiserum (dilution 1:lOOO) and goat anti-rabbit IgG conjugated with alkaline phosphatase (Sigma). Powdered milk was used instead of BSA-gelatin as a nonspecific blocking agent in these experiments. Nucleotide binding using [~I-~*P]GTP (3000 Cilmmol; Amersham) was analyzed on nitrocellulose filters as described by Lapetina and Reep (1987). Metabolic Labeling and lmmunoprecipitation Wild-type and sec6-4 cells were grown at 25°C in minimal medium supplemented with 2% glucose and 100 KM ammonium sulfate. Forty OD,$, units of cells were sedimented and resuspended in 30 ml of minimal medium containing 0.2% glucose and 50 KM ammonium sulfate. After 30 min at 37°C cells were pelleted and resuspended in 1 ml of the same medium without sulfate and containing 1 mCi [%]methionine (1477 Cilmmol; Amersham). After 1 min or 5 min at 37% cells were washed twice at CC in 50 m M Tris (pH 7.5) containing 10 m M azide, IO m M cold methionine, and 10 pglml of cycloheximide. Cells were then spheroplasted with zymolase in 1.4 M sorbitol, 50 m M Tris (pH 7.5) in the presence of azide, cold methionine, and cycloheximide, lysed, and fractionated as described above. For chase experiments, cells were washed after labeling in 50 m M T& (pH 7.5) containing 10 m M cold methionine and incubated for 1 hr at 37% in minimal medium supplemented with 100 nM ammonium sulfate and 10 m M cold methionine. In some experiments, cells were lysed with glass beads and separated into crude membrane and soluble fractions as described by Fujiyama and Tamanoi (1986). For immunoprecipitation, aliquots of radiolabeled samples boiled in 1% SDS were diluted IO-fold with 1 ml of phosphate-buffered saline (PBS) containing 2% Triton X-100 and centrifuged for 15 min in an Eppendorf tube at 4°C. The supernatant (0.9 ml) was removed, and preimmune or immune antiSec4p antiserum (about 2 PI per 1 ODsg9 U cell equivalent) was added for an overnight incubation at 4%. Protein A Sepharose (Sigma) was then added (80 nl of a 10% solution per 2 ~1 of serum) for 90 min at 4OC. The immune complexes were washed twice with 1 ml of 2 M urea, 0.2 M NaCI, 1% Triton X-100, 0.1 M Tris (pH 7.6) and twice with 1 ml of 1% b-mercaptoethanol in water, and the pellets were subjected to SDS gel electrophoresis. Fluorography was performed by soaking the gels twice in 10% methanol, followed by a 1 hr incubation of 0.3 M salicylate (pH 7.0)

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