Streamlined Architecture and Glycosylphosphatidylinositol-dependent Trafficking in the Early Secretory Pathway of African Trypanosomes

The variant surface glycoprotein (VSG) of bloodstream form Trypanosoma brucei (Tb) is a critical virulence factor. The VSG glycosylphosphatidylinositol (GPI)-anchor strongly influences passage through the early secretory pathway. Using a dominant-negative mutation of TbSar1, we show that endoplasmic reticulum (ER) exit of secretory cargo in trypanosomes is dependent on the coat protein complex II (COPII) machinery. Trypanosomes have two orthologues each of the Sec23 and Sec24 COPII subunits, which form specific heterodimeric pairs: TbSec23.1/TbSec24.2 and TbSec23.2/TbSec24.1. RNA interference silencing of each subunit is lethal but has minimal effects on trafficking of soluble and transmembrane proteins. However, silencing of the TbSec23.2/TbSec24.1 pair selectively impairs ER exit of GPI-anchored cargo. All four subunits colocalize to one or two ER exit sites (ERES), in close alignment with the postnuclear flagellar adherence zone (FAZ), and closely juxtaposed to corresponding Golgi clusters. These ERES are nucleated on the FAZ-associated ER. The Golgi matrix protein Tb Golgi reassembly stacking protein defines a region between the ERES and Golgi, suggesting a possible structural role in the ERES:Golgi junction. Our results confirm a selective mechanism for GPI-anchored cargo loading into COPII vesicles and a remarkable degree of streamlining in the early secretory pathway. This unusual architecture probably maximizes efficiency of VSG transport and fidelity in organellar segregation during cytokinesis.     

Sec16 defines endoplasmic reticulum exit sites and is required for secretory cargo export in mammalian cells

The selective export of proteins and lipids from the endoplasmic reticulum (ER) is mediated by the coat protein complex II (COPII) that assembles onto the ER membrane. In higher eukaryotes, COPII proteins assemble at discrete sites on the membrane known as ER exit sites (ERES). Here, we identify Sec16 as the protein that defines ERES in mammalian cells. Sec16 localizes to ERES independent of Sec23/24 and Sec13/31. Overexpression, and to a lesser extent, small interfering RNA depletion of Sec16, both inhibit ER-to-Golgi transport suggesting that Sec16 is required in stoichiometric amounts. Sar1 activity is required to maintain the localization of Sec16 at discrete locations on the ER membrane, probably through preventing its dissociation. Our data suggest that Sar1-GTP-dependent assembly of Sec16 on the ER membrane forms an organized scaffold defining an ERES.     

Role of Rab1b in COPII dynamics and function

In eukaryotic cells, proteins destined for secretion are translocated into the endoplasmic reticulum (ER) and packaged into so-called COPII-coated vesicles. In the ER exit sites (ERES), COPII has the capacity of deforming the lipid bilayer, where it modulates the selective sorting and concentration of cargo proteins. In this study, we analyze the involvement of Rab1b in COPII dynamics and function by expressing either the Rab1b negative-mutant (Rab1N121I) or the Rab1b GTP restricted mutant (Rab1Q67L), or performing short interference RNA-based knockdown. We show that Rab1b interacts with the COPII components Sec23, Sec24 and Sec31 and that Rab1b inhibition changes the COPII phenotype. FRAP assays reveal that Rab1b modulates COPII association/dissociation kinetics at the ERES interface. Furthermore, Rab1b inhibition delays cargo sorting at the ER exit sites. We postulate that Rab1b is a key regulatory component of COPII dynamics and function.     

De novo formation of plant endoplasmic reticulum export sites is membrane cargo induced and signal mediated

The plant endoplasmic reticulum (ER) contains functionally distinct subdomains at which cargo molecules are packed into transport carriers. To study these ER export sites (ERES), we used tobacco (Nicotiana tabacum) leaf epidermis as a model system and tested whether increased cargo dosage leads to their de novo formation. We have followed the subcellular distribution of the known ERES marker based on a yellow fluorescent protein (YFP) fusion of the Sec24 COPII coat component (YFP-Sec24), which, differently from the previously described ERES marker, tobacco Sar1-YFP, is visibly recruited at ERES in both the presence and absence of overexpressed membrane cargo. This allowed us to quantify variation in the ERES number and in the recruitment of Sec24 to ERES upon expression of cargo. We show that increased synthesis of membrane cargo leads to an increase in the number of ERES and induces the recruitment of Sec24 to these ER subdomains. Soluble proteins that are passively secreted were found to leave the ER with no apparent up-regulation of either the ERES number or the COPII marker, showing that bulk flow transport has spare capacity in vivo. However, de novo ERES formation, as well as increased recruitment of Sec24 to ERES, was found to be dependent on the presence of the diacidic ER export motif in the cytosolic domain of the membrane cargo. Our data suggest that the plant ER can adapt to a sudden increase in membrane cargo-stimulated secretory activity by signal-mediated recruitment of COPII machinery onto existing ERES, accompanied by de novo generation of new ERES.     

Adaptation of endoplasmic reticulum exit sites to acute and chronic increases in cargo load

The biogenesis of endoplasmic reticulum (ER) exit sites (ERES) involves the formation of phosphatidylinositol-4 phosphate (PI4) and Sec16, but it is entirely unknown how ERES adapt to variations in cargo load. Here, we studied acute and chronic adaptive responses of ERES to an increase in cargo load for ER export. The acute response (within minutes) to increased cargo load stimulated ERES fusion events, leading to larger but less ERES. Silencing either PI4-kinase IIIα (PI4K-IIIα) or Sec16 inhibited the acute response. Overexpression of secretory cargo for 24 h induced the unfolded protein response (UPR), upregulated COPII, and the cells formed more ERES. This chronic response was insensitive to silencing PI4K-IIIα, but was abrogated by silencing Sec16. The UPR was required as the chronic response was absent in cells lacking inositol-requiring protein 1. Mathematical model simulations further support the notion that increasing ERES number together with COPII levels is an efficient way to enhance the secretory flux. These results indicate that chronic and acute increases in cargo load are handled differentially by ERES and are regulated by different factors.     

Dynamic organization of COPII coat proteins at endoplasmic reticulum export sites in plant cells

Protein export from the endoplasmic reticulum (ER) is mediated by the accumulation of COPII proteins such as Sar1, Sec23/24 and Sec13/31 at specialized ER export sites (ERES). Although the distribution of COPII components in mammalian and yeast systems is established, a unified model of ERES dynamics has yet to be presented in plants. To investigate this, we have followed the dynamics of fluorescent fusions to inner and outer components of the coat, AtSec24 and AtSec13, in three different plant model systems: tobacco and Arabidopsis leaf epidermis, as well as tobacco BY-2 suspension cells. In leaves, AtSec24 accumulated at Golgi-associated ERES, whereas AtSec13 showed higher levels of cytosolic staining compared with AtSec24. However, in BY-2 cells, both AtSec13 and AtSec24 labelled Golgi-associated ERES, along with AtSec24. To correlate the distribution of the COPII coat with the dynamics of organelle movement, quantitative live-cell imaging analyses demonstrated that AtSec24 and AtSec13 maintained a constant association with Golgi-associated ERES, irrespective of their velocity. However, recruitment of AtSec24 and AtSec13 to ERES, as well as the number of ERES marked by these proteins, was influenced by export of membrane cargo proteins from the ER to the Golgi. Additionally, the increased availability of AtSec24 affected the distribution of AtSec13, inducing recruitment of this outer COPII coat component to ERES. These results provide a model that, in plants, protein export from the ER occurs via sequential recruitment of inner and outer COPII components to form transport intermediates at mobile, Golgi-associated ERES.     

Mammalian Sec16/p250 plays a role in membrane traffic from the endoplasmic reticulum

Coat protein complex II (COPII)-coated vesicles/carriers, which mediate export of proteins from the endoplasmic reticulum (ER), are formed at special ER subdomains in mammals, termed ER exit sites or transitional ER. The COPII coat consists of a small GTPase, Sar1, and two protein complexes, Sec23-Sec24 and Sec13-Sec31. Sec23-Sec24 and Sec13-Sec31 appear to constitute the inner and the outermost layers of the COPII coat, respectively. We previously isolated two mammalian proteins (p125 and p250) that bind to Sec23. p125 was found to be a mammalian-specific, phospholipase A(1)-like protein that participates in the organization of ER exit sites. Here we show that p250 is encoded by the KIAA0310 clone and has sequence similarity to yeast Sec16 protein. Although KIAA0310p was found to be localized at ER exit sites, subcellular fractionation revealed its predominant presence in the cytosol. Cytosolic KIAA0310p was recruited to ER membranes in a manner dependent on Sar1. Depletion of KIAA0310p mildly caused disorganization of ER exit sites and delayed protein transport from the ER, suggesting its implication in membrane traffic out of the ER. Overexpression of KIAA0310p affected ER exit sites in a manner different from that of p125. Binding experiments suggested that KIAA0310p interacts with both the inner and the outermost layer coat complexes, whereas p125 binds principally to the inner layer complex. Our results suggest that KIAA0310p, a mammalian homologue of yeast Sec16, builds up ER exit sites in cooperation with p125 and plays a role in membrane traffic from the ER.     

Endoplasmic reticulum export sites and Golgi bodies behave as single mobile secretory units in plant cells

In contrast with animals, plant cells contain multiple mobile Golgi stacks distributed over the entire cytoplasm. However, the distribution and dynamics of protein export sites on the plant endoplasmic reticulum (ER) surface have yet to be characterized. A widely accepted model for ER-to-Golgi transport is based on the sequential action of COPII and COPI coat complexes. The COPII complex assembles by the ordered recruitment of cytosolic components on the ER membrane. Here, we have visualized two early components of the COPII machinery, the small GTPase Sar1p and its GTP exchanging factor Sec12p in live tobacco (Nicotiana tabacum) leaf epidermal cells. By in vivo confocal laser scanning microscopy and fluorescence recovery after photobleaching experiments, we show that Sar1p cycles on mobile punctate structures that track with the Golgi bodies in close proximity but contain regions that are physically separated from the Golgi bodies. By contrast, Sec12p is uniformly distributed along the ER network and does not accumulate in these structures, consistent with the fact that Sec12p does not become part of a COPII vesicle. We propose that punctate accumulation of Sar1p represents ER export sites (ERES). The sites may represent a combination of Sar1p-coated ER membranes, nascent COPII membranes, and COPII vectors in transit, which have yet to lose their coats. ERES can be induced by overproducing Golgi membrane proteins but not soluble bulk-flow cargos. Few punctate Sar1p loci were observed that are independent of Golgi bodies, and these may be nascent ERES. The vast majority of ERES form secretory units that move along the surface of the ER together with the Golgi bodies, but movement does not influence the rate of cargo transport between these two organelles. Moreover, we could demonstrate using the drug brefeldin A that formation of ERES is strictly dependent on a functional retrograde transport route from the Golgi apparatus.     

Adaptor functions of the Ca2+-binding protein ALG-2 in protein transport from the endoplasmic reticulum

Apoptosis-linked gene 2 (ALG-2) is a Ca²⁺-binding protein with five repetitive EF-hand motifs, named penta-EF-hand (PEF) domain. It interacts with various target proteins and functions as a Ca²⁺-dependent adaptor in diverse cellular activities. In the cytoplasm, ALG-2 is predominantly localized to a specialized region of the endoplasmic reticulum (ER), called the ER exit site (ERES), through its interaction with Sec31A. Sec31A is an outer coat protein of coat protein complex II (COPII) and is recruited from the cytosol to the ERES to form COPII-coated transport vesicles. I will overview current knowledge of the physiological significance of ALG-2 in regulating ERES localization of Sec31A and the following adaptor functions of ALG-2, including bridging Sec31A and annexin A11 to stabilize Sec31A at the ERES, polymerizing the Trk-fused gene (TFG) product, and linking MAPK1-interacting and spindle stabilizing (MISS)-like (MISSL) and microtubule-associated protein 1B (MAP1B) to promote anterograde transport from the ER.     

A New Role for Annexin A11 in the Early Secretory Pathway via Stabilizing Sec31A Protein at the Endoplasmic Reticulum Exit Sites (ERES)

Exit of cargo molecules from the endoplasmic reticulum (ER) for transport to the Golgi is the initial step in intracellular vesicular trafficking. The coat protein complex II (COPII) machinery is recruited to specialized regions of the ER, called ER exit sites (ERES), where it plays a central role in the early secretory pathway. It has been known for more than two decades that calcium is an essential factor in vesicle trafficking from the ER to Golgi apparatus. However, the role of calcium in the early secretory pathway is complicated and poorly understood. We and others previously identified Sec31A, an outer cage component of COPII, as an interacting protein for the penta-EF-hand calcium-binding protein ALG-2. In this study, we show that another calcium-binding protein, annexin A11 (AnxA11), physically associates with Sec31A by the adaptor function of ALG-2. Depletion of AnxA11 or ALG-2 decreases the population of Sec31A that is stably associated with the ERES and causes scattering of juxtanuclear ERES to the cell periphery. The synchronous ER-to-Golgi transport of transmembrane cargoes is accelerated in AnxA11- or ALG-2-knockdown cells. These findings suggest that AnxA11 maintains architectural and functional features of the ERES by coordinating with ALG-2 to stabilize Sec31A at the ERES.     

Dynamics of COPII vesicles and the Golgi apparatus in cultured Nicotiana tabacum BY-2 cells provides evidence for transient association of Golgi stacks with endoplasmic reticulum exit sites

Despite the ubiquitous presence of the COPI, COPII, and clathrin vesicle budding machineries in all eukaryotes, the organization of the secretory pathway in plants differs significantly from that in yeast and mammalian cells. Mobile Golgi stacks and the lack of both transitional endoplasmic reticulum (ER) and a distinct ER-to-Golgi intermediate compartment are the most prominent distinguishing morphological features of the early secretory pathway in plants. Although the formation of COPI vesicles at periphery of Golgi cisternae has been demonstrated in plants, exit from the ER has been difficult to visualize, and the spatial relationship of this event is now a matter of controversy. Using tobacco (Nicotiana tabacum) BY-2 cells, which represent a highly active secretory system, we have used two approaches to investigate the location and dynamics of COPII binding to the ER and the relationship of these ER exit sites (ERES) to the Golgi apparatus. On the one hand, we have identified endogenous COPII using affinity purified antisera generated against selected COPII-coat proteins (Sar1, Sec13, and Sec23); on the other hand, we have prepared a BY-2 cell line expressing Sec13:green fluorescent protein (GFP) to perform live cell imaging with red fluorescent protein-labeled ER or Golgi stacks. COPII binding to the ER in BY-2 cells is visualized as fluorescent punctate structures uniformly distributed over the surface of the ER, both after antibody staining as well as by Sec13:GFP expression. These structures are smaller and greatly outnumber the Golgi stacks. They are stationary, but have an extremely short half-life (<10 s). Without correlative imaging data on the export of membrane or lumenal ER cargo it was not possible to equate unequivocally these COPII binding loci with ERES. When a GDP-fixed Sar1 mutant is expressed, ER export is blocked and the visualization of COPII binding is perturbed. On the other hand, when secretion is inhibited by brefeldin A, COPII binding sites on the ER remain visible even after the Golgi apparatus has been lost. Live cell imaging in a confocal laser scanning microscope equipped with spinning disk optics allowed us to investigate the relationship between mobile Golgi stacks and COPII binding sites. As they move, Golgi stacks temporarily associated with COPII binding sites at their rims. Golgi stacks were visualized with their peripheries partially or fully occupied with COPII. In the latter case, Golgi stacks had the appearance of a COPII halo. Slow moving Golgi stacks tended to have more peripheral COPII than faster moving ones. However, some stationary Golgi stacks entirely lacking COPII were also observed. Our results indicate that, in a cell type with highly mobile Golgi stacks like tobacco BY-2, the Golgi apparatus is not continually linked to a single ERES. By contrast, Golgi stacks associate intermittently and sometimes concurrently with several ERES as they move.     

COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER–ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins’ role in ER-to-Golgi transport.     

Endoplasmic reticulum exit of a secretory glycoprotein in the absence of sec24p family proteins in yeast

Glycoproteins exit the endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae in coat protein complex II (COPII) coated vesicles. The coat consists of the essential proteins Sec23p, Sec24p, Sec13p, Sec31p, Sar1p and Sec16p. Sec24p and its two nonessential homologues Sfb2p and Sfb3p have been suggested to serve in cargo selection. Using temperature-sensitive sec24-1 mutants, we showed previously that a secretory glycoprotein, Hsp150, does not require functional Sec24p for ER exit. Deletion of SFB2, SFB3 or both from wild type or the deletion of SFB2 from sec24-1 cells did not affect Hsp150 transport. SFB3 deletion has been reported to be lethal in sec24-1. However, here we constructed a sec24-1 Deltasfb3 and a sec24-1 Deltasfb2 Deltasfb3 strain and show that Hsp150 was secreted slowly in both. Turning off the SEC24 gene did not inhibit Hsp150 secretion either, and the lack of SEC24 expression in a Deltasfb2 Deltasfb3 deletant still allowed some secretion. The sec24-1 Deltasfb2 Deltasfb3 mutant grew slower than sec24-1. The cells were irregularly shaped, budded from random sites and contained proliferated ER at permissive temperature. At restrictive temperature, the ER formed carmellae-like proliferations. Our data indicate that ER exit may occur in vesicles lacking a full complement of Sec23p/24p and Sec13p/31p, demonstrating diversity in the composition of the COPII coat.     

Insights into structural and regulatory roles of Sec16 in COPII vesicle formation at ER exit sites

COPII-coated buds are formed at endoplasmic reticulum exit sites (ERES) to mediate ER-to-Golgi transport. Sec16 is an essential factor in ERES formation, as well as in COPII-mediated traffic in vivo. Sec16 interacts with multiple COPII proteins, although the functional significance of these interactions remains unknown. Here we present evidence that full-length Sec16 plays an important role in regulating Sar1 GTPase activity at the late steps of COPII vesicle formation. We show that Sec16 interacts with Sec23 and Sar1 through its C-terminal conserved region and hinders the ability of Sec31 to stimulate Sec23 GAP activity toward Sar1. We also find that purified Sec16 alone can self-assemble into homo-oligomeric complexes on a planar lipid membrane. These features ensure prolonged COPII coat association within a preformed Sec16 cluster, which may lead to the formation of ERES. Our results indicate a mechanistic relationship between COPII coat assembly and ERES formation.     

Biogenesis of cytoplasmic membranous vesicles for plant potyvirus replication occurs at endoplasmic reticulum exit sites in a COPI- and COPII-dependent manner

Single-stranded positive-sense RNA viruses induce the biogenesis of cytoplasmic membranous vesicles, where viral replication takes place. However, the mechanism underlying this characteristic vesicular proliferation remains poorly understood. Previously, a 6-kDa potyvirus membrane protein (6K) was shown to interact with the endoplasmic reticulum (ER) and to induce the formation of the membranous vesicles. In this study, the involvement of the early secretory pathway in the formation of the 6K-induced vesicles was investigated in planta. By means of live-cell imaging, it was found that the 6K protein was predominantly colocalized with Sar1, Sec23, and Sec24, which are known markers of ER exit sites (ERES). The localization of 6K at ERES was prevented by the coexpression of a dominant-negative mutant of Sar1 that disables the COPII activity or by the coexpression of a mutant of Arf1 that disrupts the COPI complex. The secretion of a soluble secretory marker targeting the apoplast was arrested at the level of the ER in cells overexpressing 6K or infected by a potyvirus. This blockage of protein trafficking out of the ER by 6K and the distribution of 6K toward the ERES may account for the aggregation of the 6K-bound vesicles. Finally, virus infection was reduced when the accumulation of 6K at ERES was inhibited by impairing either the COPI or COPII complex. Taken together, these results imply that the cellular COPI and COPII coating machineries are involved in the biogenesis of the potyvirus 6K vesicles at the ERES for viral-genome replication.     

The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.     

Sorting of GPI-anchored proteins into ER exit sites by p24 proteins is dependent on remodeled GPI

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a posttranslational modification occurring in the endoplasmic reticulum (ER). After GPI attachment, proteins are transported by coat protein complex II (COPII)-coated vesicles from the ER. Because GPI-anchored proteins (GPI-APs) are localized in the lumen, they cannot interact with cytosolic COPII components directly. Receptors that link GPI-APs to COPII are thought to be involved in efficient packaging of GPI-APs into vesicles; however, mechanisms of GPI-AP sorting are not well understood. Here we describe two remodeling reactions for GPI anchors, mediated by PGAP1 and PGAP5, which were required for sorting of GPI-APs to ER exit sites. The p24 family of proteins recognized the remodeled GPI-APs and sorted them into COPII vesicles. Association of p24 proteins with GPI-APs was pH dependent, which suggests that they bind in the ER and dissociate in post-ER acidic compartments. Our results indicate that p24 complexes act as cargo receptors for correctly remodeled GPI-APs to be sorted into COPII vesicles.     

The yeast p24 complex regulates GPI-anchored protein transport and quality control by monitoring anchor remodeling

Glycosylphosphatidylinositol (GPI)-anchored proteins are secretory proteins that are attached to the cell surface of eukaryotic cells by a glycolipid moiety. Once GPI anchoring has occurred in the lumen of the endoplasmic reticulum (ER), the structure of the lipid part on the GPI anchor undergoes a remodeling process prior to ER exit. In this study, we provide evidence suggesting that the yeast p24 complex, through binding specifically to GPI-anchored proteins in an anchor-dependent manner, plays a dual role in their selective trafficking. First, the p24 complex promotes efficient ER exit of remodeled GPI-anchored proteins after concentration by connecting them with the COPII coat and thus facilitates their incorporation into vesicles. Second, it retrieves escaped, unremodeled GPI-anchored proteins from the Golgi to the ER in COPI vesicles. Therefore the p24 complex, by sensing the status of the GPI anchor, regulates GPI-anchored protein intracellular transport and coordinates this with correct anchor remodeling.     

Ceramide biosynthesis is required for the formation of the oligomeric H+-ATPase Pma1p in the yeast endoplasmic reticulum

The yeast plasma membrane H(+)-ATPase Pma1p is one of the most abundant proteins to traverse the secretory pathway. Newly synthesized Pma1p exits the endoplasmic reticulum (ER) via COPII-coated vesicles bound for the Golgi. Unlike most secreted proteins, efficient incorporation of Pma1p into COPII vesicles requires the Sec24p homolog Lst1p, suggesting a unique role for Lst1p in ER export. Vesicles formed with mixed Sec24p-Lst1p coats are larger than those with Sec24p alone. Here, we examined the relationship between Pma1p biosynthesis and the requirement for this novel coat subunit. We show that Pma1p forms a large oligomeric complex of >1 MDa in the ER, which is packaged into COPII vesicles. Furthermore, oligomerization of Pma1p is linked to membrane lipid composition; Pma1p is rendered monomeric in cells depleted of ceramide, suggesting that association with lipid rafts may influence oligomerization. Surprisingly, monomeric Pma1p present in ceramide-deficient membranes can be exported from the ER in COPII vesicles in a reaction that is stimulated by Lst1p. We suggest that Lst1p directly conveys Pma1p into a COPII vesicle and that the larger size of mixed Sec24pLst1p COPII vesicles is not essential to the packaging of large oligomeric complexes.     

Selective protein exit from yeast endoplasmic reticulum in absence of functional COPII coat component Sec13p

Sec13p has been thought to be an essential component of the COPII coat, required for exit of proteins from the yeast endoplasmic reticulum (ER). We show herein that normal function of Sec13p was not required for ER exit of the Hsp150 glycoprotein. Hsp150 was secreted to the medium under restrictive conditions in a sec13-1 mutant. The COPII components Sec23p and Sec31p and the GTP/GDP exchange factor Sec12p were required in functional form for secretion of Hsp150. Hsp150 leaves the ER in the absence of retrograde COPI traffic, and the responsible determinant is a peptide repeated 11 times in the middle of the Hsp150 sequence. Herein, we localized the sorting determinant for Sec13p-independent ER exit to the C-terminal domain. Sec13p-dependent invertase left the ER in the absence of normal Sec13p function, when fused to the C-terminal domain of Hsp150, demonstrating that this domain contained an active mediator of Sec13p-independent secretion. Thus, Hsp150 harbors two different signatures that regulate its ER exit. Our data show that transport vesicles lacking functional Sec13p can carry out ER-to-Golgi transport, but select only specific cargo protein(s) for ER exit.