The Transitional ER Localization Mechanism of Pichia pastoris Sec12

Developmental Cell, Vol. 6, 649–659, May, 2004, Copyright 2004 by Cell Press

The Transitional ER Localization Mechanism of Pichia pastoris Sec12 Jon Soderholm, Dibyendu Bhattacharyya, Daniel Strongin, Vida Markovitz, Pamela L. Connerly, Catherine A. Reinke, and Benjamin S. Glick* Department of Molecular Genetics and Cell Biology The University of Chicago 920 East 58th Street Chicago, Illinois 60637

Summary COPII vesicles assemble at ER subdomains called transitional ER (tER) sites, but the mechanism that generates tER sites is unknown. To study tER biogenesis, we analyzed the transmembrane protein Sec12, which initiates COPII vesicle formation. Sec12 is concentrated at discrete tER sites in the budding yeast Pichia pastoris. We find that P. pastoris Sec12 exchanges rapidly between tER sites and the general ER. The tER localization of Sec12 is saturable and is mediated by interaction of the Sec12 cytosolic domain with a partner component. This interaction apparently requires oligomerization of the Sec12 lumenal domain. Redistribution of P. pastoris Sec12 to the general ER does not perturb the localization of downstream tER components, suggesting that Sec12 and other COPII proteins associate with a tER scaffold. These results provide evidence that tER sites form by a network of dynamic associations at the cytosolic face of the ER. Introduction Newly synthesized proteins exit the ER in COPII transport vesicles (Antonny and Schekman, 2001; Barlowe, 2002). In most eukaryotes, COPII coat proteins and nascent COPII vesicles are concentrated at tER sites, which are ribosome-free ER subdomains approximately 0.5 ␮m in diameter (Palade, 1975; Bannykh and Balch, 1997). The number of tER sites per cell ranges from one in certain protists to several hundred in vertebrates (Becker and Melkonian, 1996; Hammond and Glick, 2000a). While the COPII assembly process is well characterized, little is known about how COPII vesicle formation is restricted to tER sites. We are addressing this question using the budding yeasts Pichia pastoris and Saccharomyces cerevisiae. P. pastoris has a “conventional” secretory apparatus: a typical cell contains two to five tER sites, each of which is juxtaposed to a polarized Golgi stack (Rossanese et al., 1999; Mogelsvang et al., 2003). By contrast, S. cerevisiae has an unusual secretory apparatus: COPII vesicles bud from the entire ER, and individual Golgi cisternae are scattered throughout the cytoplasm (Franzusoff et al., 1991; Preuss et al., 1992; Rossanese et al., 1999). We have proposed that the tER is the birthplace of the Golgi, and that analyzing tER organization in budding yeasts will shed light on *Correspondence: [email protected]

the mechanisms that determine Golgi structure (Glick, 2002). In previous work, we labeled tER sites in P. pastoris by fusing green fluorescent protein (GFP) to the COPII coat protein Sec13 (Rossanese et al., 1999), and then used 4D confocal microscopy to characterize tER dynamics (Bevis et al., 2002). This approach revealed that tER sites are extremely long-lived. However, the number of tER sites per cell fluctuates because tER sites form de novo and they occasionally collide and fuse. Similar tER dynamics have been observed in mammalian cells (Hammond and Glick, 2000a; Stephens et al., 2000; Stephens, 2003). These results suggest that tER sites form by a self-organization process in which individual tER components undergo reversible, multivalent associations to create specialized patches in the ER membrane (Bevis et al., 2002). To begin testing this model, we chose to study Sec12, a transmembrane ER protein that initiates COPII vesicle formation by catalyzing guanine nucleotide exchange on the GTPase Sar1 (Nakano et al., 1988; Barlowe and Schekman, 1993). Activated Sar1-GTP binds to the ER membrane and recruits the Sec23/24 coat protein complex, which then recruits the Sec13/31 complex (Antonny and Schekman, 2001; Barlowe, 2002). The cytosolic domain of Sec12 interacts with Sar1, whereas the lumenal domain has not been ascribed a specific function. Sec12 is present throughout the ER in S. cerevisiae but is concentrated at tER sites in P. pastoris (Nishikawa and Nakano, 1993; Rossanese et al., 1999). Because Sec12 is the most upstream of the known players in the COPII assembly pathway, the tER localization of P. pastoris Sec12 is likely to involve novel interactions. One possibility is that P. pastoris Sec12 self-associates to generate tER sites. An alternative possibility is that P. pastoris Sec12 binds to a tER scaffold. Thus, analyzing the molecular basis for Sec12 localization can provide insights into the mechanism that defines the tER. Results P. pastoris Sec12 Associates Reversibly with tER Sites Our first goal was to determine whether the localization of P. pastoris Sec12 to tER sites is stable or dynamic. For this purpose, we created a P. pastoris strain containing GFP-tagged Sec12. The SEC12 gene is essential for viability in P. pastoris, as it is in S. cerevisiae (Nakano et al., 1988), so we performed a gene replacement to confirm that GFP-Sec12 was functional (see Experimental Procedures). The strain containing GFP-tagged Sec12 was examined using 4D confocal microscopy (Hammond and Glick, 2000b). GFP-Sec12 was concentrated at punctate tER sites that were juxtaposed to Golgi structures marked with Sec7-DsRed, although low concentrations of GFP-Sec12 were also detectable in the general ER (Figure 1A and data not shown). GFPSec12-labeled tER sites were long-lived structures that exhibited fusion and apparent de novo formation, as

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Figure 1. P. pastoris Sec12 Rapidly Exchanges between tER Sites and the General ER A strain was constructed in which the endogenous P. pastoris SEC12 gene was deleted and GFP-SEC12 was expressed from the SEC12 promoter (see Experimental Procedures). We found that a Sec12 fusion to one or three copies of the standard enhanced GFP (EGFP; Cormack et al., 1996) yielded virtually no fluorescence, but a fusion to three tandem copies of an alternative GFP variant (sGFP; Kahana et al., 1998) yielded a good signal. (A) 4D confocal microscopy was used to examine the dynamics of GFP-Sec12 in living P. pastoris cells. Shown are selected panels from Supplemental Movie S1 (http://www.developmentalcell.com/cgi/content/full/6/5/649/DC1), with times indicated in min:s format. The arrowhead points to a GFP-Sec12-labeled tER site that was bleached and then allowed to recover fluorescence. Scale bar, 2 ␮m. (B) Fluorescence recovery after photobleaching was quantified for the tER site marked in (A). The horizontal axis indicates the time in seconds after the photobleaching, and the vertical axis indicates fluorescence intensity in arbitrary units. The half-time of fluorescence recovery for this tER site was approximately 60 s. Bleaching of this tER site resulted in a loss of approximately 65% of the total cellular fluorescence, and as a result, the tER site recovered only about 35% of its original fluorescence.

previously observed using the COPII coat protein Sec13-GFP (Bevis et al., 2002). To determine whether GFP-Sec12 exchanged between tER sites and the general ER, we performed fluorescence recovery after photobleaching (FRAP) analysis. When individual tER sites were bleached, the fluorescence recovered rapidly, with half-times in the range of 15 to 90 s (Figures 1A and 1B). Thus, although the tER sites marked with GFPSec12 are very stable, individual GFP-Sec12 molecules move rapidly between tER sites and the general ER. tER Localization of P. pastoris Sec12 Requires a Partner Component The dynamic tER localization of P. pastoris Sec12 could occur by either of two mechanisms. First, Sec12 might self-associate to form patches in the ER membrane. Second, Sec12 might associate with tER sites by interacting with a partner component. To distinguish between these mechanisms, we overexpressed Sec12 in P. pastoris cells. If Sec12 self-associates, then overexpression should generate large patches of concentrated Sec12. By contrast, if the tER localization of Sec12 requires binding to a partner component, then overexpressing Sec12 should saturate the binding sites, and the excess Sec12 molecules should be found in the general ER. For this experiment, endogenous P. pastoris Sec12 was marked with a GluGlu epitope tag (EEEEYMPME; Grussenmeyer et al., 1985) immediately preceding the C-terminal HDEL tetrapeptide (Payne et al., 2000). We found that the acidic GluGlu tag preserved the tER localization of P. pastoris Sec12, whereas less acidic epitope tags such as hemagglutinin (YPYDVPDYA) and c-myc (EQKLISEEDL) perturbed tER localization to varying degrees (data not shown). In the cells containing GluGlutagged Sec12 (Sec12-GG), untagged Sec12 was overexpressed from the methanol-inducible AOX1 promoter (Koutz et al., 1989; Sears et al., 1998). As shown in Figure 2, Sec12-GG was concentrated at tER sites before methanol induction (Figure 2D). After 4 hr of methanol induction, Sec12-GG was still concentrated at tER sites,

which were of normal size (Figure 2E). After 8 hr of methanol induction, Sec12-GG was delocalized throughout the ER as indicated by prominent staining of the nuclear envelope and cytoplasmic ER tubules (Figure 2F). In the absence of Sec12 overexpression, growth on methanol did not cause delocalization of Sec12-GG (Figures 2A–2C). To verify the overexpression, endogenous Sec12 was left untagged and Sec12GG was overexpressed (Figures 2G–2I). Immunoblotting (data not shown) revealed that methanol-induced Sec12-GG overexpression was moderate (ⵑ12-fold) after 4 hr and strong (ⵑ190-fold) after 8 hr. Overexpression of Sec12 had no significant effect either on cell growth or on tER organization as visualized using Sec13GFP (data not shown). Thus, strong overexpression of Sec12 resulted in a specific redistribution of this protein throughout the ER, indicating that the tER localization of Sec12 is saturable. Saturation probably occurred even with moderate overexpression of Sec12, but because much more membrane is present in the general ER than in tER sites, the delocalized Sec12 only became visible when the Sec12 concentration in the general ER approached the concentration in tER sites. These data suggest that P. pastoris Sec12 localizes to tER sites by interacting with a partner component. Unlike P. pastoris Sec12, S. cerevisiae Sec12 is present throughout the entire ER (Nishikawa and Nakano, 1993; Rossanese et al., 1999). Presumably, the P. pastoris partner component that mediates tER localization of Sec12 either is absent in S. cerevisiae, or does not perform the same Sec12 clustering function in S. cerevisiae. It follows that if P. pastoris Sec12 were expressed in S. cerevisiae, it should be found in the general ER. This prediction was tested by expressing P. pastoris Sec12-GG in a sec12⌬ strain of S. cerevisiae (Figure 3A). P. pastoris Sec12-GG is functional in S. cerevisiae because it rescues the lethal sec12⌬ phenotype. We initially tried expressing P. pastoris Sec12-GG from a centromeric plasmid but found that the protein levels were too low to detect, presumably because the heterologous P. pastoris gene is expressed inefficiently from

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Figure 2. The Association of P. pastoris Sec12 with tER Sites Is Saturable P. pastoris cells were grown to log phase in SYG, washed, then transferred to SYM for various times to induce expression from the AOX1 promoter. The cells were processed for immunofluorescence with anti-GluGlu antibody either immediately following the switch to SYM (t ⫽ 0 hr), after 4 hr of growth in SYM (t ⫽ 4 hr), or after 8 hr of growth in SYM (t ⫽ 8 hr). Sec12-GG, GluGlu-tagged Sec12 in which the C-terminal sequence EGENINHDEL was replaced by EGENINEEEEYMPMEEALHDEL. OEx, overexpression. Scale bar, 2 ␮m. (A–C) The chromosomal copy of SEC12 was replaced with SEC12-GG under control of the endogenous SEC12 promoter. These cells were transformed with the parental AOX1 promoter vector pIB4. (D–F) The chromosomal copy of SEC12 was replaced with SEC12-GG under control of the endogenous SEC12 promoter. These cells were transformed with a derivative of pIB4 encoding untagged P. pastoris Sec12 under control of the AOX1 promoter. (G–I) P. pastoris cells containing a wild-type chromosomal copy of SEC12 were transformed with a vector encoding Sec12-GG under control of the AOX1 promoter. All of the cells were photographed with an identical exposure time, except that the exposure time for (I) was shorter because the fluorescence signal was very strong.

the S. cerevisiae SEC12 promoter. Therefore, the P. pastoris SEC12-GG gene was placed on a 2 ␮m plasmid. Although this plasmid conferred high-level expression on average, the amount of Sec12-GG varied dramatically from cell to cell, allowing us to assess the effects of different expression levels within a single culture. The upper panels in Figure 3A show S. cerevisiae cells containing amounts of P. pastoris Sec12-GG ranging from barely detectable to high. In all cases, P. pastoris Sec12 displayed a general ER pattern indistinguishable from that of S. cerevisiae Sec12-GG (Figure 3A). We conclude that tER localization of P. pastoris Sec12 does not reflect an intrinsic self-association of this protein, but instead reflects binding of Sec12 to a tER-localized partner component. Multiple Regions of P. pastoris Sec12 Are Required for tER Localization The binding of P. pastoris Sec12 to the tER-localized partner component probably involves a specific region of Sec12. This region can potentially be identified

through a comparison with S. cerevisiae Sec12, assuming that S. cerevisiae Sec12 is not recognized by the tER localization system of P. pastoris. To test this assumption, we expressed S. cerevisiae Sec12-GG in P. pastoris. For unknown reasons, S. cerevisiae Sec12 cannot functionally replace P. pastoris Sec12 even at high expression levels (data not shown), so S. cerevisiae Sec12-GG was expressed in a P. pastoris strain that also contained endogenous untagged P. pastoris Sec12. Moderate expression of S. cerevisiae Sec12-GG yielded a general ER pattern, whereas expression of P. pastoris Sec12-GG under the same conditions yielded a tER pattern (Figure 3B). This result supports the idea that a tER binding region is present in P. pastoris Sec12 but not in S. cerevisiae Sec12. A comparison between the two Sec12 proteins revealed pronounced differences that could account for the different localization patterns (Figure 4A). Even though the cytosolic domain of Sec12 is the enzymatically active part of the protein, the Sec12 cytosolic domains from P. pastoris and S. cerevisiae share only 36%

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Figure 3. P. pastoris Sec12 Contains a tER Localization Signal that Functions in P. pastoris but Not in S. cerevisiae (A) Either P. pastoris Sec12-GG or S. cerevisiae Sec12-GG was expressed from the S. cerevisiae SEC12 promoter in an S. cerevisiae strain lacking the endogenous chromosomal copy of SEC12. The tagged proteins were visualized by immunofluorescence. In each case, cells are shown expressing low (left panels), moderate (middle panels), or high (right panels) levels of Sec12-GG. The contrast of these images was adjusted to facilitate comparison. (B) Either S. cerevisiae Sec12-GG or P. pastoris Sec12-GG was expressed from the AOX1 promoter in a P. pastoris strain that also contained untagged endogenous Sec12. Cells were processed for immunofluorescence after 4 hr of growth in SYM. As shown in Figure 2, after 4 hr of AOX1-driven expression, Sec12 protein levels are too low to saturate tER localization. Nevertheless, in this and subsequent experiments employing the AOX1 promoter, we carefully examined cells exhibiting varying levels of expression to ensure that a general ER pattern was not due to overexpression. Scale bar, 2 ␮m.

identity (Payne et al., 2000). There is no significant similarity between the lumenal domains of the two Sec12 proteins. The lumenal domain of P. pastoris Sec12 has the following features that are not present in S. cerevisiae Sec12: the C-terminal tetrapeptide is HDEL, which likely acts as a Golgi-to-ER retrieval signal (Pelham, 1995); preceding the HDEL sequence is an extremely acidic segment (labeled “A” in Figure 4A); upstream of the acidic segment is a large central segment (labeled “C” in Figure 4A); and proximal to the ER membrane is a basic segment (labeled “B” in Figure 4A). To identify the portions of P. pastoris Sec12 that are important for tER localization, we made a set of chimeric constructs in which the cytosolic, transmembrane, or lumenal domain of P. pastoris Sec12 was replaced with the corresponding domain from S. cerevisiae Sec12. When the transmembrane domain was replaced, the chimeric protein still localized to tER sites (Figure 4C, middle panel). When the cytosolic domain was replaced, the chimeric protein localized to the general ER (Figure 4C, left panel). The same pattern of general ER staining was seen when the cytosolic domain was removed (Figure 4B, left panel). (These constructs lacking the cytosolic domain of P. pastoris Sec12 could not support growth of P. pastoris, and were therefore expressed in cells that also contained a mutant P. pastoris Sec12 protein lacking the lumenal domain, as described below.) Our results strongly suggest that the cytosolic domain of P. pastoris Sec12 is essential for tER localization. Surprisingly, the lumenal domain is also essential

for tER localization. When the lumenal domain of P. pastoris Sec12 was replaced (Figure 4C, right panel) or removed (Figure 4B, right panel), the mutant proteins localized to the general ER. In these cases, the mutant Sec12 proteins represented all of the Sec12 in the cells, yet the cells showed no obvious growth defect. Thus, the lumenal domain of P. pastoris Sec12 is dispensable for the function of Sec12 in COPII vesicle assembly but is required for tER localization. By systematically removing individual segments of the P. pastoris Sec12 lumenal domain, we found that either the basic segment alone or the acidic segment alone was sufficient for tER localization (when linked to the transmembrane and cytosolic domains) (Figure 5). The combined results indicate that tER localization of P. pastoris Sec12 requires both the cytosolic domain and either of two segments of the lumenal domain. tER Localization of P. pastoris Sec12 Involves Oligomerization of the Lumenal Domain How can we account for the multiplicity of tER localization determinants in P. pastoris Sec12? We speculated that either the lumenal or the cytosolic domain of Sec12 binds to the tER-localized partner component, while the other domain oligomerizes as a prerequisite for this binding. To test whether oligomerization is important for localizing Sec12 to tER sites, we replaced either the lumenal or the cytosolic domain of Sec12 with a 33amino acid leucine zipper from the Gcn4 transcription factor (O’Shea et al., 1989). The Gcn4 leucine zipper

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Figure 4. tER Localization of P. pastoris Sec12 Requires Both the Cytosolic and Lumenal Domains Chimeric or truncated forms of P. pastoris Sec12 were expressed in P. pastoris cells and examined by immunofluorescence. Each construct included a GluGlu tag, either at the C terminus or just upstream of the HDEL tetrapeptide (see Figure 2 legend). (A) Schematic diagram of Sec12 from P. pastoris (“P.p.,” orange) and S. cerevisiae (“S.c.,” blue). The lumenal domain of P. pastoris Sec12 includes a basic segment (“B;” residues 360– 598), a central segment (“C;” residues 599–950), an acidic segment (“A;” residues 951–1034), and a C-terminal HDEL tetrapeptide. (B) Left panel: the cytosolic domain of P. pastoris Sec12 (residues 2–328) was removed. This construct was expressed from the SEC12 promoter in a P. pastoris strain containing a truncated Sec12 that lacked the lumenal domain. Right panel: the lumenal domain of P. pastoris Sec12 (residues 367–1038) was removed. This construct was made by gene replacement at the SEC12 locus, and to boost expression to detectable levels, a second copy of the same truncated gene was expressed from the AOX1 promoter with 4 hr of induction in SYM. Note that the nuclei tend to be smaller in methanol-grown P. pastoris cells than in glucose-grown cells. Scale bar, 2 ␮m. (C) Left panel: the cytosolic domain of P. pastoris Sec12 (residues 1–332) was replaced with the cytosolic domain of S. cerevisiae Sec12 (residues 1–349). This construct was expressed from the AOX1 promoter with 4 hr of induction in SYM, in a P. pastoris strain containing a truncated Sec12 that lacked the lumenal domain. Middle panel: the transmembrane domain of P. pastoris Sec12 (residues 338–359) was replaced with the transmembrane domain of S. cerevisiae Sec12 (residues 355–373). This construct was expressed from the SEC12 promoter in a sec12⌬ strain of P. pastoris. Right panel: the lumenal domain of P. pastoris Sec12 (residues 360–1038) was replaced with the lumenal domain of S. cerevisiae Sec12 (residues 374–471). This construct was made by gene replacement at the SEC12 locus, and to boost expression to detectable levels, a second copy of the same chimeric gene was expressed from the AOX1 promoter with 4 hr of induction in SYM.

(Figure 6A) has been extensively analyzed in vitro and in vivo and is known to form a stable parallel coiled coil (O’Shea et al., 1989; Hu et al., 1990). When the lumenal domain of P. pastoris Sec12 was replaced with the Gcn4 leucine zipper, the resulting chimeric protein (Sec12-LZ) localized to tER sites (Figure 6B, left panel), as confirmed by colocalization with Sec13-GFP (data not shown). tER localization was abolished by mutating leucine-19 of the Gcn4 sequence to proline (Figure 6B, right panel); this mutation was previously shown to disrupt the leucine zipper (Hu et al., 1990). When the cytosolic domain of P. pastoris Sec12 was replaced with the Gcn4 leucine zipper, the resulting chimeric protein (LZ-Sec12) was found in the general ER (Figure 6C). These results suggest that tER localization of P. pastoris Sec12 requires a specific structure within the cytosolic domain, and also requires oligomerization of the lumenal domain. According to this model, either the basic or the acidic segment of the lumenal domain is sufficient to promote oligomerization. To assay for oligomerization, we performed a yeast two-hybrid analysis (James et al., 1996) using the basic segment of the P. pastoris Sec12 lumenal domain as both “bait” and “prey.” The basic segment was chosen for the two-hybrid test because it lacks cysteine residues and N-linked glycosylation sites, and because it is sufficient to confer tER localization (see Figure 5). Growth was seen on media lacking either histidine or adenine (Figure 6D, lane 2), suggesting that the basic segment can oligomerize.

tER Localization of Sec12 Is Not Required for tER or Golgi Organization Given that Sec12 initiates the COPII vesicle budding pathway, it seemed reasonable that Sec12 localization might determine the position of nascent COPII vesicles. This possibility was tested by examining tER sites in a P. pastoris strain that contained a delocalized version of Sec12. We used a strain in which the lumenal domain of P. pastoris Sec12 had been replaced with that of S. cerevisiae Sec12 (see Figure 4C, right panel). In this strain, the localization of Sec13-GFP (Figure 7A) and the dynamics of Sec13-GFP-labeled tER sites (data not shown; see Bevis et al., 2002) were essentially normal. Because the primary function of Sec12 is to recruit Sar1 to the ER membrane, we also examined the distribution of Sar1, which had not previously been visualized in P. pastoris. Sar1 colocalizes with Sec13-GFP in P. pastoris cells, regardless of whether Sec12 is concentrated at tER sites (Figure 7A). Delocalization of Sec12 also had no discernible effect on the apposition of tER sites and Golgi stacks, as judged by immunofluorescence using the tER marker Sec13-GFP and the Golgi marker Och1-HA (Figure 7B) (Rossanese et al., 1999). This result was confirmed by electron microscopy (Figure 7C): in cells expressing either wild-type or delocalized Sec12, Golgi stacks were seen next to vesiculating regions of the nuclear envelope or cortical ER. Compared to the wild-type strain, the strain containing delocalized Sec12 did show an increase in the number of both tER sites and Golgi stacks per cell, but this increase was slight (less than 2-fold on

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Figure 5. The Lumenal Domain of P. pastoris Sec12 Contains Redundant tER Localization Signals (A) The endogenous SEC12 locus in P. pastoris was replaced with a series of constructs encoding the indicated deletions of segments of the Sec12 lumenal domain (see Figure 4A for segment descriptions). Each construct included a GluGlu tag, either at the C terminus or just upstream of the HDEL tetrapeptide (see Figure 2 legend). For the ⌬B,A,HDEL construct and the ⌬B,C construct, expression was boosted to detectable levels by expressing a second copy of the same mutant gene from the AOX1 promoter with 4 hr of induction in SYM. The localization of the mutant proteins was analyzed by immunofluorescence. (B) Representative immunofluorescence images are shown for the ⌬C,A,HDEL construct (left panel), the ⌬B,A,HDEL construct (middle panel), and the ⌬B,C construct (right panel). The punctate structures seen with the ⌬C,A,HDEL and ⌬B,C constructs also labeled with Sec13-GFP, confirming that these structures are tER sites (not shown). Scale bar, 2 ␮m.

average). Thus, delocalization of P. pastoris Sec12 does not significantly change the organization of the tERGolgi system. Discussion tER sites were identified many years ago (Palade, 1975; Whaley, 1975), but the molecular architecture of these structures has remained mysterious. As an entry point into this problem, we chose to study P. pastoris Sec12. FRAP analysis revealed that a GFP-tagged version of Sec12 exchanges rapidly between tER sites and the general ER (Figure 1). This observation supports a model in which tER sites are generated by a network of weak, reversible interactions (Bevis et al., 2002). The question then becomes: which specific interactions are critical for tER site formation? One idea was that Sec12 itself might define tER sites. Sec12 would self-associate to form patches, which would recruit Sar1, which in turn would recruit COPII coat proteins. This scenario was

consistent with current knowledge of the COPII assembly pathway (Antonny and Schekman, 2001; Barlowe, 2002). An alternative idea was that Sec12 might bind to unidentified components of a tER scaffold (Rossanese et al., 1999). In this scenario, COPII proteins that function downstream of Sec12 would localize to tER sites either through the action of Sec12, or else through direct binding to the tER scaffold. Here we have addressed these various possibilities. If P. pastoris Sec12 self-associated to form patches, then overexpression of Sec12 should generate larger patches. In fact, when we overexpressed P. pastoris Sec12 in P. pastoris cells, the excess Sec12 was found in the general ER (Figure 2). A similar general ER pattern was seen when P. pastoris Sec12 was expressed in S. cerevisiae (Figure 3). These results demonstrate that P. pastoris Sec12 does not have an intrinsic ability to cluster into patches. Instead, the clustering of P. pastoris Sec12 seems to require binding to a saturable, tERlocalized partner component.

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Figure 6. Oligomerization of the Lumenal Domain Promotes tER Localization of P. pastoris Sec12 (A) Shown at the top is the sequence of the Gcn4 leucine zipper. The schematic diagrams represent mutant versions of P. pastoris Sec12 in which the leucine zipper (small white rectangle) replaced either the lumenal domain in the construct Sec12-LZ or the cytosolic domain in the construct LZ-Sec12. In Sec12-LZ(L19P), the leucine in the Gcn4 sequence at position 19 (arrow) was changed to a proline. Each of these proteins had a C-terminal GluGlu tag. (B) Immunofluorescence of cells containing Sec12-LZ (left panel) or Sec12-LZ(L19P) (right panel). Each construct was made by gene replacement, and to boost expression to detectable levels, a second copy of the same mutant gene was expressed from the AOX1 promoter with 4 hr of induction in SYM. (C) Immunofluorescence of cells containing LZ-Sec12. This construct was expressed from the SEC12 promoter in a P. pastoris strain containing a truncated Sec12 that lacked the lumenal domain. Scale bar, 2 ␮m. (D) Yeast two-hybrid analysis using a URA3 “bait” vector and a LEU2 “prey” vector (James et al., 1996). Lane 1: empty bait and prey vectors. Lane 2: Bait and prey vectors both expressing fusions to the basic segment of the P. pastoris Sec12 lumenal domain. Lane 3: Empty prey vector with a bait vector expressing a Sec12 basic segment fusion. Lane 4: Empty bait vector with a prey vector expressing a Sec12 basic segment fusion. Cells growing on minimal medium lacking leucine (leu) and uracil (ura) were patched either on the same medium (left panel) or on media that also lacked histidine (his; middle panel) or adenine (ade; right panel), and were incubated at 30⬚C for 3 days.

Which features of P. pastoris Sec12 are responsible for tER localization? One possibility is that Sec12 localizes by binding to Sar1, which has been proposed to organize tER sites by linking cargo sorting to recruitment of the COPII coat (Aridor et al., 2001). However, expression of S. cerevisiae Sec12 in P. pastoris resulted in general ER localization (Figure 3), and a chimeric Sec12 was found throughout the ER even though Sar1 was concentrated at tER sites (Figures 4 and 7), indicating that Sar1 binding is not sufficient to localize Sec12 to tER sites. The implication is that P. pastoris Sec12 contains a specific tER localization determinant. To identify this determinant, we generated a set of truncated, internally

deleted, and chimeric Sec12 proteins (Figures 4 and 5). Unexpectedly, these experiments defined three distinct regions of P. pastoris Sec12 that are involved in tER localization: the cytosolic domain, a membrane-proximal basic segment of the lumenal domain, and a highly acidic C-terminal segment of the lumenal domain. We propose that the Sec12 cytosolic domain binds to the tER-localized partner component. This conclusion is based on the finding that tER localization of Sec12 was lost when the cytosolic domain was deleted (Figure 4) or was replaced either with a dimeric coiled coil (Figure 6), with mouse dihydrofolate reductase (J.S., unpublished data), or with the cytosolic domain of S. cerevisiae

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Figure 7. Delocalization of Sec12 Does Not Perturb the Localization of Downstream tER or Golgi Components tER and Golgi organization was examined by immunofluorescence and electron microscopy in P. pastoris strains containing either tER-localized wild-type Sec12, or a delocalized chimeric Sec12 in which the lumenal domain had been replaced with that of S. cerevisiae Sec12 (see Figure 4C). This chimeric Sec12 was expressed from the endogenous SEC12 promoter. The strains used in (A) and (B) also expressed Sec13-GFP. (A) Double label immunofluorescence of the tER markers Sar1 and Sec13-GFP in cells containing either wild-type Sec12 (top panels) or delocalized chimeric Sec12 (bottom panels). Scale bar, 2 ␮m. (B) Double label immunofluorescence of the tER marker Sec13-GFP and the Golgi marker Och1-HA in cells containing either wild-type Sec12 (top panels) or delocalized chimeric Sec12 (bottom panels). (C) Representative electron micrographs of P. pastoris cells containing either wild-type Sec12 (left panel) or delocalized chimeric Sec12 (right panel). Electron microscopy was performed as previously described (Rossanese et al., 1999). N, nucleus. Scale bars, 0.25 ␮m.

Sec12 (Figure 4). The role of the Sec12 lumenal domain in tER localization is probably to form dimers or higherorder oligomers. This conclusion is based on the finding that tER localization of Sec12 was retained when the lumenal domain was replaced with a peptide that dimerizes (Figure 6). As judged by immunofluorescence and two-hybrid analysis, oligomerization of the lumenal domain can apparently be effected by either the membrane-proximal basic segment or the C-terminal acidic segment (Figures 5 and 6). The combined results suggest that the cytosolic domain of P. pastoris Sec12 binds to a tER-localized partner component in a manner that requires oligomerization mediated by the lumenal domain. Interestingly, the mammalian ER membrane protein CLIMP-63 is excluded from the nuclear envelope due to oligomerization of its lumenal domain (Klopfenstein et al., 2001), although the localization mechanism for CLIMP-63 may be quite different from that of P. pastoris Sec12. Important future steps will include identifying the tER-localized partner component that

binds the Sec12 cytosolic domain, and quantifying the oligomerization properties of the Sec12 lumenal domain. Three additional points merit further study. The first point is that in gene replacement tests, P. pastoris Sec12 can substitute for S. cerevisiae Sec12 (Figure 3; Payne et al., 2000) but not vice versa. A trivial possibility is that S. cerevisiae Sec12 might not fold correctly in P. pastoris or might not recognize P. pastoris Sar1. A more attractive explanation is that P. pastoris Sec12 might perform a second function, apart from tER localization, that cannot be performed by S. cerevisiae Sec12. Perhaps this second function is carried out in S. cerevisiae by Sed4, a Sec12 homolog found only in S. cerevisiae and very closely related species (Hardwick et al., 1992; Payne et al., 2000). The second point is the oligomerization requirement for tER localization of P. pastoris Sec12 (Figure 6). We speculate that the affinity of Sec12 for its tER-localized partner component is low, and that oligomerization enhances the avidity. Perhaps Sec12 oligomerization is readily reversible, allowing Sec12 to

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dissociate from its partner component and avoid being packaged into COPII vesicles (Barlowe et al., 1994). A test of this model will require setting up a COPII vesicle budding assay with P. pastoris components. The third point is the elaborate structure of the P. pastoris Sec12 lumenal domain. This domain probably has functions in addition to oligomerization. For example, the basic and central segments of the Sec12 lumenal domain may recruit other lumenal proteins to tER sites, and the acidic C-terminal segment of the lumenal domain may bind calcium (Nash et al., 1994; Yano and Zarain-Herzberg, 1994). To put the analysis of Sec12 in a functional context, we asked whether a concentrated localization of Sec12 is needed to generate tER sites. This experiment involved comparing P. pastoris strains that contained either wild-type Sec12 or a chimeric Sec12 that was delocalized throughout the ER. In these two strains, the organization of the tER-Golgi system was very similar (Figure 7). This result implies the existence of a tER scaffold that does not include Sec12 as an essential component. Interestingly, mammalian Sec12 is found throughout the ER rather than being concentrated at tER sites (Weissman et al., 2001), consistent with the idea that Sec12 is not generally needed to establish tER organization. Moreover, Sec12 can function without being concentrated at sites of COPII vesicle formation. One possible interpretation is that Sec12 can recruit Sar1-GTP throughout the ER, and the Sar1-GTP then diffuses along the ER surface until it is captured by a nascent COPII vesicle. The tER localization of P. pastoris Sec12 may have evolved to make this capture of Sar1GTP more efficient. As described above, the tER localization of P. pastoris Sec12 may also allow the lumenal domain of this protein to recruit additional components to tER sites. Such recruitment probably does not occur in S. cerevisiae or mammalian cells, in which the Sec12 proteins localize to the general ER and contain small lumenal domains (Nakano et al., 1988; Weissman et al., 2001). The combined data suggest that Sec12 action and tER organization are mechanistically similar in P. pastoris and in mammalian cells, but that P. pastoris Sec12 has additional specialized features. If Sec12 does not define tER sites, then what does? A reasonable candidate is the tER-localized partner component that binds the cytosolic domain of P. pastoris Sec12. This partner component may also bind other COPII proteins. In other words, we postulate the existence of a tER scaffold located at the cytosolic surface of the ER. This tER scaffold seems to include the large peripheral ER membrane protein Sec16 (P.L.C., unpublished data). S. cerevisiae Sec16 interacts with several COPII proteins and modulates COPII vesicle formation (Gimeno et al., 1996; Shaywitz et al., 1997; Supek et al., 2002). Although S. cerevisiae Sec16 has not been observed to bind Sec12, it does bind the Sec12 homolog Sed4 (Gimeno et al., 1995), suggesting that a Sec16Sec12 interaction might occur in P. pastoris. Our current work is aimed at testing whether the properties of Sec16 in P. pastoris and S. cerevisiae can account for the different distributions of COPII proteins in these two yeasts.

Experimental Procedures Strains and Plasmids All strains used were derivatives of the P. pastoris strain PPY12 (his4 arg4; Gould et al., 1992) or the S. cerevisiae strain JK9-3d (leu2-3,112 ura3-52 trp1 his4 rme1; Kunz et al., 1993). P. pastoris cells were transformed by electroporation (Sears et al., 1998), and S. cerevisiae cells were transformed using lithium acetate (Gietz and Woods, 2002). Unless otherwise indicated, yeast were grown in rich glucose medium (YPD) or minimal glucose medium (SD) (Sherman, 1991). Diploid P. pastoris strains were sporulated and subjected to random spore analysis as described (Gould et al., 1992), except that 100% ethanol was used instead of ether to kill vegetative cells. Diploid S. cerevisiae strains were sporulated and subjected to tetrad dissection (Sherman, 1991). The P. pastoris diploid PPY12D was constructed as follows. We generated PPY12 haploid strains carrying episomal plasmids (Cregg et al., 1985) marked with either the S. cerevisiae HIS4 gene or the S. cerevisiae ARG4 gene. A His⫹ transformant was then mated with an Arg⫹ transformant as described (Gould et al., 1992), except that starting cultures were grown overnight in SD medium lacking histidine or arginine rather than in YPD. Diploids resulting from this mating were selected on SD plates lacking both histidine and arginine. Individual diploid clones were then grown overnight in liquid YPD to allow spontaneous loss of the plasmids. These cultures were diluted and spread on YPD plates. A clone that grew on YPD but not on SD plates lacking histidine or arginine was designated PPY12D (his4/his4 arg4/arg4). P. pastoris strains expressing Sec13-GFP and Och1-HA were generated as previously described (Rossanese et al., 1999). The procedures used here to manipulate the SEC12 genes are summarized below. Plasmids containing part or all of the P. pastoris SEC12 gene exhibit varying degrees of toxicity in E. coli, so many of the DNA cloning steps required extra care to maintain the plasmids. In particular, carbenicillin was used instead of ampicillin, and bacterial transformants were streaked on plates before being grown in liquid culture. To express a wild-type or modified SEC12 gene in P. pastoris cells that also contained a functional endogenous version of SEC12, the second copy SEC12 gene was integrated at the HIS4 locus under control of either the P. pastoris SEC12 promoter or the methanolinducible AOX1 promoter using the vectors pIB1 and pIB4, respectively (Sears et al., 1998). For methanol induction, cells were grown to log phase in the minimal glucose medium SYG (Sears et al., 1998). The SYG was then filtered away, and cells were washed once in the minimal methanol medium SYM and then resuspended in SYM. Growth was allowed to continue in SYM for 0, 4, or 8 hr before the cells were processed for immunofluorescence. Two strategies were used to express a modified SEC12 gene in P. pastoris as the only copy of SEC12. The first strategy was to create an ARG4 plasmid containing a modified 3⬘ fragment of SEC12, then perform gene replacement at the SEC12 locus by homologous recombination in haploid PPY12 cells (Rossanese et al., 1999). The second strategy was to introduce a modified SEC12 gene at the HIS4 locus of a diploid containing one disrupted copy of SEC12 (see below), then sporulate to generate a haploid containing the modified gene as the only copy of SEC12. For this second strategy, an EcoRI-BamHI fragment containing the entire P. pastoris SEC12 gene (Payne et al., 2000) was subcloned into pUC19 (Yanisch-Perron et al., 1985). An NcoI site was incorporated at the start codon to yield pJS1. (Introduction of this NcoI site changed the second amino acid of Sec12 from Met to Val, but control experiments showed that this change did not alter Sec12 expression.) The SEC12 gene was then excised from pJS1 with EcoRI and PstI and subcloned between the corresponding sites in pIB1 to yield pJS2. Derivatives of pJS2 were used to express modified versions of SEC12 in P. pastoris from the native SEC12 promoter. For example, to facilitate detection of Sec12 in P. pastoris, pJS2 was mutagenized to encode a GluGlu epitope tag immediately preceding the C-terminal HDEL sequence. When we needed to boost the expression of a modified SEC12 gene in P. pastoris using the AOX1 promoter, a derivative of pJS2 containing the modified SEC12 gene was digested with NcoI, blunted, then digested with PstI, and the liberated fragment was subcloned into pIB4 that had been digested with Asp718, blunted, and then digested with PstI.

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To disrupt one copy of P. pastoris SEC12 in PPY12D, an NcoIAvrII fragment containing most of the SEC12 open reading frame was excised from pJS1 and replaced with a HpaI fragment containing the S. cerevisiae ARG4 gene. The resulting 3.6 kb disruption cassette was excised with EcoRI and PstI and transformed into PPY12D, and correct integration at the SEC12 locus was confirmed by PCR. As a control, one copy of AOX1 was replaced in PPY12D with S. cerevisiae ARG4 by transformation with a 6.3 kb EcoRIPvuII fragment from pYM17 (Cregg and Madden, 1987). AOX1 is dispensable for growth on glucose medium (Koutz et al., 1989), and random spore analysis revealed that aox1⌬::ARG4 haploids were viable whereas sec12⌬::ARG4 haploids were inviable, confirming that SEC12 is essential. The SEC12/sec12⌬ diploid was transformed with derivatives of pJS2 and sporulated, yielding Arg⫹ His⫹ spores that contained the pJS2-derived gene as the only functional copy of SEC12. To express a tagged SEC12 gene in S. cerevisiae as the only copy of SEC12, we first disrupted one copy of SEC12 in a JK9-3d diploid using the KanMX gene cassette (Wach et al., 1994). This disrupted diploid was transformed with a derivative of the 2 ␮m URA3 plasmid YEplac195 (Gietz and Sugino, 1988) containing the S. cerevisiae SEC12 promoter followed by the SEC12-GG gene from either S. cerevisiae or P. pastoris. The resulting strain was then sporulated to generate a kanamycin-resistant Ura⫹ haploid. Fluorescence Microscopy Immunofluorescence microscopy was performed as previously described (Rossanese et al., 1999), except that to visualize Sar1, cells were fixed for 2 hr on ice in buffer containing 2% formaldehyde. Anti-GluGlu monoclonal antibody (Covance, Berkeley, CA) was used at 20 ␮g/ml. Anti-HA monoclonal antibody (clone 16B12; Covance), anti-c-myc monoclonal antibody (clone 9E10; Roche, Indianapolis, IN), and anti-GFP monoclonal antibody (mixture from clones 7.1 and 13.1; Roche) were used at 5 ␮g/ml. To produce the anti-Sar1 antibody, an intronless copy of S. cerevisiae SAR1 was inserted into pQE-80L (Qiagen, Valencia, CA) and expressed in DH10B E. coli cells. Hexahistidine-tagged Sar1 was purified using Ni2⫹-NTA-agarose and sent to Zymed Laboratories (San Francisco, CA) for production of rabbit polyclonal antibodies. The resulting antiserum recognized S. cerevisiae Sar1 and P. pastoris Sar1 in crude yeast lysates, or in lysates of E. coli cells expressing either Sar1 protein. The antiserum was affinity purified (Pringle et al., 1991) using a strip of polyvinylidine fluoride (PVDF) membrane containing recombinant S. cerevisiae Sar1, and the purified antibody was diluted 1:1000 for immunofluorescence. As a specificity control, preincubation of the diluted antibody with 0.7 mg/ml of either S. cerevisiae Sar1 or P. pastoris Sar1 resulted in complete quenching of the Sar1 signal from P. pastoris cells. For 4D confocal microscopy of P. pastoris cells on a Zeiss LSM 510 confocal microscope (Hammond and Glick, 2000b; Bevis et al., 2002), stacks of 16 optical slices spaced 0.38 ␮m apart were collected at 1 s intervals. FRAP analysis was performed by using the Edit Bleach feature of the Zeiss software to bleach a single tER site with 100% laser power. The bleach period extended through the central 14 of the 16 optical slices in the stack. Immunoblotting For immunoblotting of P. pastoris Sec12, protein was extracted from cells by adjusting the culture medium to 10% trichloroacetic acid, incubating at 50⬚C for 5 min, chilling on ice for 5 min, centrifuging for 5 min at 2000 ⫻ g, and resuspending in SDS-PAGE sample buffer supplemented with 50 mM Na⫹-PIPES (pH 7.5). Samples were heated at 50⬚C in sample buffer because higher temperatures caused Sec12 to aggregate. Each lane of the gel contained ⵑ50 ␮g of cellular protein extract. The separated proteins were transferred to PVDF membranes, and tagged Sec12 was detected with the SuperSignal West Femto kit (Pierce, Rockford, IL). To quantify Sec12-GG overexpression, serial dilutions of the extracts from overexpressing cells were compared with extracts from cells expressing endogenous levels of Sec12-GG. Acknowledgments This work was supported by NIH grant GM61156-01 to B.S.G. and by NIH training grant 5-20942 to J.S., D.S., P.L.C., and C.A.R. We

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