Cargo Capture and Bulk Flow in the Early Secretory Pathway.

Transport of newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi complex is highly selective. As a general rule, such transport is limited to soluble and membrane-associated secretory proteins that have reached properly folded and assembled conformations. To secure the efficiency, fidelity, and control of this crucial transport step, cells use a combination of mechanisms. The mechanisms are based on selective retention of proteins in the ER to prevent uptake into transport vesicles, on selective capture of proteins in COPII carrier vesicles, on inclusion of proteins in these vesicles by default as part of fluid and membrane bulk flow, and on selective retrieval of proteins from post-ER compartments by retrograde vesicle transport.

Protein sorting receptors in the early secretory pathway.

Estimates based on proteomic analyses indicate that a third of translated proteins in eukaryotic genomes enter the secretory pathway. After folding and assembly of nascent secretory proteins in the endoplasmic reticulum (ER), the coat protein complex II (COPII) selects folded cargo for export in membrane-bound vesicles. To accommodate the great diversity in secretory cargo, protein sorting receptors are required in a number of instances for efficient ER export. These transmembrane sorting receptors couple specific secretory cargo to COPII through interactions with both cargo and coat subunits. After incorporation into COPII transport vesicles, protein sorting receptors release bound cargo in pre-Golgi or Golgi compartments, and receptors are then recycled back to the ER for additional rounds of cargo export. Distinct types of protein sorting receptors that recognize carbohydrate and/or polypeptide signals in secretory cargo have been characterized. Our current understanding of the molecular mechanisms underlying cargo receptor function are described.

The C-terminus of the cargo receptor Erv14 affects COPII vesicle formation and cargo delivery.

The endoplasmic reticulum (ER) is the start site of the secretory pathway, where newly synthesized secreted and membrane proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Little is known about how post-translational modification events regulate packaging of cargo into COPII vesicles. The Saccharomyces cerevisiae protein Erv14, also known as cornichon, belongs to a conserved family of cargo receptors required for the selection and ER export of transmembrane proteins. In this work, we show the importance of a phosphorylation consensus site (S134) at the C-terminus of Erv14. Mimicking phosphorylation of S134 (S134D) prevents the incorporation of Erv14 into COPII vesicles, delays cell growth, exacerbates growth of sec mutants, modifies ER structure and affects localization of several plasma membrane transporters. In contrast, the dephosphorylated mimic (S134A) had less deleterious effects, but still modifies ER structure and slows cell growth. Our results suggest that a possible cycle of phosphorylation and dephosphorylation is important for the correct functioning of Erv14.

Organization of the ER-Golgi interface for membrane traffic control.

Coat protein complex I (COPI) and COPII are required for bidirectional membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. While these core coat machineries and other transport factors are highly conserved across species, high-resolution imaging studies indicate that the organization of the ER-Golgi interface is varied in eukaryotic cells. Regulation of COPII assembly, in some cases to manage distinct cellular cargo, is emerging as one important component in determining this structure. Comparison of the ER-Golgi interface across different systems, particularly mammalian and plant cells, reveals fundamental elements and distinct organization of this interface. A better understanding of how these interfaces are regulated to meet varying cellular secretory demands should provide key insights into the mechanisms that control efficient trafficking of proteins and lipids through the secretory pathway.

ER sheets get roughed up.

The molecular machinery that shapes the endoplasmic reticulum's (ER's) membrane into ordered networks of "smooth" tubules and "rough" sheets is poorly defined. Shibata et al. (2010) now report that sheet-inducing proteins, such as Climp-63, are enriched in the "rough" ER by their association with membrane-bound ribosomes, whereas curvature-inducing proteins localize at highly bent edges of membrane sheets.

Conserved juxtamembrane domains in the yeast golgin Coy1 drive assembly of a megadalton-sized complex and mediate binding to tethering and SNARE proteins.

The architecture and organization of the Golgi complex depend on a family of coiled-coil proteins called golgins. Golgins are thought to form extended homodimers that are C-terminally anchored to Golgi membranes, whereas their N termini extend into the cytoplasm to initiate vesicle capture. Previously, we reported that the Saccharomyces cerevisiae golgin Coy1 contributes to intra-Golgi retrograde transport and binds to the conserved oligomeric Golgi (COG) complex and multiple retrograde Golgi Q-SNAREs (where SNARE is soluble NSF-attachment protein receptor). Here, using various engineered yeast strains, membrane protein extraction and fractionation methods, and in vitro binding assays, we mapped the Coy1 regions responsible for these activities. We also report that Coy1 assembles into a megadalton-size complex and that assembly of this complex depends on the most C-terminal coiled-coil and a conserved region between this coiled-coil and the transmembrane domain of Coy1. We found that this conserved region is necessary and sufficient for binding the SNARE protein Sed5 and the COG complex. Mutagenesis of conserved arginine residues within the C-terminal coiled-coil disrupted oligomerization, binding, and function of Coy1. Our findings indicate that the stable incorporation of Coy1 into a higher-order oligomer is required for its interactions and role in maintaining Golgi homeostasis. We propose that Coy1 assembles into a docking platform that directs COG-bound vesicles toward cognate SNAREs on the Golgi membrane.

Analysis of COPII Vesicles Indicates a Role for the Emp47-Ssp120 Complex in Transport of Cell Surface Glycoproteins.

Coat protein complex II (COPII) vesicle formation at the endoplasmic reticulum (ER) transports nascent secretory proteins forward to the Golgi complex. To further define the machinery that packages secretory cargo and targets vesicles to Golgi membranes, we performed a comprehensive proteomic analysis of purified COPII vesicles. In addition to previously known proteins, we identified new vesicle proteins including Coy1, Sly41 and Ssp120, which were efficiently packaged into COPII vesicles for trafficking between the ER and Golgi compartments. Further characterization of the putative calcium-binding Ssp120 protein revealed a tight association with Emp47 and in emp47Δ cells Ssp120 was mislocalized and secreted. Genetic analyses demonstrated that EMP47 and SSP120 display identical synthetic positive interactions with IRE1 and synthetic negative interactions with genes involved in cell wall assembly. Our findings support a model in which the Emp47-Ssp120 complex functions in transport of plasma membrane glycoproteins through the early secretory pathway.

Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes play essential roles in catalyzing intracellular membrane fusion events although the assembly pathway and molecular arrangement of SNARE complexes in membrane fusion reactions are not well understood. Here we monitored interactions of the R-SNARE protein Sec22 through a cysteine scanning approach and detected efficient formation of cross-linked Sec22 homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. When specific Sec22 cysteine derivatives are present on both donor COPII vesicles and acceptor Golgi membranes, the formation of disulfide cross-links provide clear readouts on trans- and cis-SNARE arrangements during this fusion event. The Sec22 transmembrane domain was required for efficient homodimer formation and for membrane fusion suggesting a functional role for Sec22 homodimers. We propose that Sec22 homodimers promote assembly of higher-order SNARE complexes to catalyze membrane fusion. Sec22 is also reported to function in macroautophagy and in formation of endoplasmic reticulum-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes.

The Dsl1 protein tethering complex is a resident endoplasmic reticulum complex, which interacts with five soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors (SNAREs): implications for fusion and fusion regulation.

Retrograde vesicular transport from the Golgi to the ER requires the Dsl1 tethering complex, which consists of the three subunits Dsl1, Dsl3, and Tip20. It forms a stable complex with the SNAREs Ufe1, Use1, and Sec20 to mediate fusion of COPI vesicles with the endoplasmic reticulum. Here, we analyze molecular interactions between five SNAREs of the ER (Ufe1, Use1, Sec20, Sec22, and Ykt6) and the Dsl1 complex in vitro and in vivo. Of the two R-SNAREs, Sec22 is preferred over Ykt6 in the Dsl-SNARE complex. The NSF homolog Sec18 can displace Ykt6 but not Sec22, suggesting a regulatory function for Ykt6. In addition, our data also reveal that subunits of the Dsl1 complex (Dsl1, Dsl3, and Tip20), as well as the SNAREs Ufe1 and Sec20, are ER-resident proteins that do not seem to move into COPII vesicles. Our data support a model, in which a tethering complex is stabilized at the organelle membrane by binding to SNAREs, recognizes the incoming vesicle via its coat and then promotes its SNARE-mediated fusion.

The yeast orthologue of GRASP65 forms a complex with a coiled-coil protein that contributes to ER to Golgi traffic.

The mammalian Golgi protein GRASP65 is required in assays that reconstitute cisternal stacking and vesicle tethering. Attached to membranes by an N-terminal myristoyl group, it recruits the coiled-coil protein GM130. The relevance of this system to budding yeasts has been unclear, as they lack an obvious orthologue of GM130, and their only GRASP65 relative (Grh1) lacks a myristoylation site and has even been suggested to act in a mitotic checkpoint. In this study, we show that Grh1 has an N-terminal amphipathic helix that is N-terminally acetylated and mediates association with the cis-Golgi. We find that Grh1 forms a complex with a previously uncharacterized coiled-coil protein, Ydl099w (Bug1). In addition, Grh1 interacts with the Sec23/24 component of the COPII coat. Neither Grh1 nor Bug1 are essential for growth, but biochemical assays and genetic interactions with known mediators of vesicle tethering (Uso1 and Ypt1) suggest that the Grh1-Bug1 complex contributes to a redundant network of interactions that mediates consumption of COPII vesicles and formation of the cis-Golgi.

Erv26p-dependent export of alkaline phosphatase from the ER requires lumenal domain recognition.

Active sorting at the endoplasmic reticulum (ER) drives efficient export of fully folded secretory proteins into coat protein complex II (COPII) vesicles, whereas ER-resident and misfolded proteins are retained and/or degraded. A number of secretory proteins depend upon polytopic cargo receptors for linkage to the COPII coat and ER export. However, the mechanism by which cargo receptors recognize transport-competent cargo is poorly understood. Here we examine the sorting determinants required for export of yeast alkaline phosphatase (ALP) by its cargo receptor Erv26p. Analyses of ALP chimeras and mutants indicated that Erv26p recognizes sorting information in the lumenal domain of ALP. This lumenal domain sorting signal must be positioned near the inner leaflet of the ER membrane for Erv26p-dependent export. Moreover, only assembled ALP dimers were efficiently recognized by Erv26p while an ALP mutant blocked in dimer assembly failed to exit the ER and was subjected to ER-associated degradation. These results further refine sorting information for ER export of ALP and show that recognition of folded cargo by export receptors contributes to strict ER quality control.

Yet1p and Yet3p, the yeast homologs of BAP29 and BAP31, interact with the endoplasmic reticulum translocation apparatus and are required for inositol prototrophy.

The mammalian B-cell receptor-associated proteins of 29 and 31 kDa (BAP29 and BAP31) are conserved integral membrane proteins that have reported roles in endoplasmic reticulum (ER) quality control, ER export of secretory cargo, and programmed cell death. In this study we investigated the yeast homologs of BAP29 and BAP31, known as Yet1p and Yet3p, to gain insight on cellular function. We found that Yet1p forms a complex with Yet3p (Yet complex) and that complex assembly was important for subunit stability and proper ER localization. The Yet complex was not efficiently packaged into ER-derived COPII vesicles and therefore does not appear to act as an ER export receptor. Instead, a fraction of the Yet complex was detected in association with the ER translocation apparatus (Sec complex). Specific mutations in the Sec complex or Yet complex influenced these interactions. Moreover, associations between the Yet complex and Sec complex were increased by ER stress and diminished when protein translocation substrates were depleted. Surprisingly, yet1Delta and yet3Delta mutant strains displayed inositol starvation-related growth defects. In accord with the biochemical data, these growth defects were exacerbated by a combination of certain mutations in the Sec complex with yet1Delta or yet3Delta mutations. We propose a model for the Yet-Sec complex interaction that places Yet1p and Yet3p at the translocation pore to manage biogenesis of specific transmembrane secretory proteins.

COPII-dependent transport from the endoplasmic reticulum.

The coat protein complex II (COPII) forms transport vesicles from the endoplasmic reticulum and segregates biosynthetic cargo from ER-resident proteins. Recent high-resolution structural studies on individual COPII subunits and on the polymerized coat reveal the molecular architecture of COPII vesicles. Other advances have shown that integral membrane accessory proteins act with the COPII coat to collect specific cargo molecules into ER-derived transport vesicles.

Sorting signals can direct receptor-mediated export of soluble proteins into COPII vesicles.

Soluble secretory proteins are first translocated across endoplasmic reticulum (ER) membranes and folded in a specialized ER luminal environment. Fully folded and assembled secretory cargo are then segregated from ER-resident proteins into COPII-derived vesicles or tubular elements for anterograde transport. Mechanisms of bulk-flow, ER-retention and receptor-mediated export have been suggested to operate during this transport step, although these mechanisms are poorly understood. In yeast, there is evidence to suggest that Erv29p functions as a transmembrane receptor for the export of certain soluble cargo proteins including glycopro-alpha-factor (gpalphaf), the precursor of alpha-factor mating pheromone. Here we identify a hydrophobic signal within the pro-region of gpalphaf that is necessary for efficient packaging into COPII vesicles and for binding to Erv29p. When fused to Kar2p, an ER-resident protein, the pro-region sorting signal was sufficient to direct Erv29p-dependent export of the fusion protein into COPII vesicles. These findings indicate that specific motifs within soluble secretory proteins function in receptor-mediated export from the ER. Moreover, positive sorting signals seem to predominate over potential ER-retention mechanisms that may operate in localizing ER-resident proteins such as Kar2p.

The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis.

alpha-Synuclein (alpha-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast alpha-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER-->Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER-->Golgi trafficking. The homologous Rab1 rescues alpha-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of alpha-syn pathobiology. In a cell-free system with purified transport factors alpha-syn inhibited ER-->Golgi trafficking in an alpha-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of alpha-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with alpha-syn and with diverse vesicle markers, suggesting that alpha-syn can impair multiple trafficking steps. Other Rabs did not ameliorate alpha-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, alpha-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.

Twenty-five years after coat protein complex II.

In 1994, a convergence of ideas and collaborative research orchestrated by Randy Schekman led to the discovery of the coat protein complex II (COPII). In this Perspective, the chain of events enabling discovery of a new vesicle coat and progress on understanding COPII budding mechanisms are considered.

Molecular dissection of the Erv41-Erv46 retrograde receptor reveals a conserved cysteine-rich region in Erv46 required for retrieval activity.

The Erv41-Erv46 complex is a conserved retrograde cargo receptor that retrieves ER resident proteins from Golgi compartments in a pH-dependent manner. Here we functionally dissect the Erv46 subunit and define an approximately 60 residue cysteine-rich region that is unique to the Erv46 family of proteins. This cysteine-rich region contains two vicinal cysteine pairs in CXXC and CCXXC configurations that are each required for retrieval activity in cells. Mutation of the individual cysteine residues produced stable Erv46 proteins that were partially reduced and form mixed-disulfide species on nonreducing gels. Conserved hydrophobic amino acids within the cysteine-rich region of Erv46 were also required for retrieval function in cells. In vitro binding experiments showed that this hydrophobic patch is required for direct cargo binding. Surprisingly, the Erv46 cysteine mutants continued to bind cargo in cell-free assays and produced an increased level of Erv46-cargo complexes in cell extracts suggesting that disulfide linkages in the cysteine-rich region perform a role in releasing bound cargo. On the basis of these findings, we propose that both pH and redox environments regulate cargo binding to a hydrophobic site within the cysteine-rich region of Erv46.

Three-dimensional structure of a COPII prebudding complex.

The coat protein complex II (COPII) catalyzes transport vesicle formation from the endoplasmic reticulum. Crystallographic analysis of a Sec23/24-Sar1 prebudding complex of COPII now provides a molecular view of this GTPase-directed coat assembly mechanism.

Signals for COPII-dependent export from the ER: what's the ticket out?

Export of many secretory proteins from the endoplasmic reticulum (ER) relies on signal-mediated sorting into ER-derived transport vesicles. Recent work on the coat protein complex II (COPII) provides new insight into the mechanisms and signals that govern this selective export process. Conserved di-acidic and di-hydrophobic motifs found in specific transmembrane cargo proteins are required for their selection into COPII-coated vesicles. These signaling elements are cytoplasmically exposed and recognized by subunits of the COPII coat. Certain soluble cargo molecules depend on receptor-like proteins for efficient ER export, although signals that direct soluble cargo into ER-derived vesicles are less defined.