Biomedical subjects

Charles Barlowe

Publications and source records attributed to Charles Barlowe.

At least 19 recordsLinked to original sources

PubMed2006

Erv26p directs pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles.

Secretory proteins are exported from the endoplasmic reticulum (ER) in transport vesicles formed by the coat protein complex II (COPII). We detected Erv26p as an integral membrane protein that was efficiently packaged into COPII vesicles and cycled between the ER and Golgi compartments. The erv26Delta mutant displayed a selective secretory defect in which the pro-form of vacuolar alkaline phosphatase (pro-ALP) accumulated in the ER, whereas other secretory proteins were transported at wild-type rates. In vitro budding experiments demonstrated that Erv26p was directly required for packaging of pro-ALP into COPII vesicles. Moreover, Erv26p was detected in a specific complex with pro-ALP when immunoprecipitated from detergent-solublized ER membranes. Based on these observations, we propose that Erv26p serves as a transmembrane adaptor to link specific secretory cargo to the COPII coat. Because ALP is a type II integral membrane protein in yeast, these findings imply that an additional class of secretory cargo relies on adaptor proteins for efficient export from the ER.

Alkaline Phosphatase↗

PubMed2006

Sel1p/Ubx2p participates in a distinct Cdc48p-dependent endoplasmic reticulum-associated degradation pathway.

The endoplasmic reticulum (ER) serves a critical role in the biogenesis of secretory proteins. Folding of nascent polypeptides occurs in the ER before anterograde transport through the secretory pathway, whereas terminally misfolded secretory proteins are recognized and eliminated by ER-associated degradation (ERAD). Here, we investigated the role of the ubiquitin regulatory X (UBX) domain-containing protein Sel1p in ER quality control and transport. Mutant sel1Delta yeast displayed a constitutively active unfolded protein response and a mildly reduced rate of secretory protein transport from the ER. Immunoisolation of Sel1p from detergent-solubilized ER microsomes revealed a protein complex containing both Cdc48p and Npl4p and suggested a direct role for Sel1p in ERAD. In cells that lack Sel1p, we observed a reduction in the level of Cdc48p bound to ER membranes and a decrease in the turnover rate of two model ERAD substrates, carboxypeptidase Y* and Ste6*. In addition, we found that Sel1p and a second UBX domain-containing protein, Shp1p, associated with Cdc48p in a mutually exclusive manner. Interestingly, the association of Sel1p with Cdc48p was regulated by ATP, while the interaction of Shp1p with Cdc48p was not influenced by ATP. Based on these findings, we conclude that Sel1p operates in the ERAD pathway by coupling Cdc48p to ER membranes and that Shp1p acts in a distinct Cdc48p-dependent protein degradation pathway.

ATP-Binding Cassette Transporters↗

PubMed2005

Cysteine-disulfide cross-linking to monitor SNARE complex assembly during endoplasmic reticulum-Golgi transport.

Assembly of cognate SNARE proteins into SNARE complexes is required for many intracellular membrane fusion reactions. However, the mechanisms that govern SNARE complex assembly and disassembly during fusion are not well understood. We have devised a new in vitro cross-linking assay to monitor SNARE complex assembly during fusion of endoplasmic reticulum (ER)-derived vesicles with Golgi-acceptor membranes. In Saccharomyces cerevisiae, anterograde ER-Golgi transport requires four SNARE proteins: Sec22p, Bos1p, Bet1p, and Sed5p. After tethering of ER-derived vesicles to Golgi-acceptor membranes, SNARE proteins are thought to assemble into a four-helix coiled-coil bundle analogous to the structurally characterized neuronal and endosomal SNARE complexes. Molecular modeling was used to generate a structure of the four-helix ER-Golgi SNARE complex. Based on this structure, cysteine residues were introduced into adjacent SNARE proteins such that disulfide bonds would form if assembled into a SNARE complex. Our initial studies focused on disulfide bond formation between the SNARE motifs of Bet1p and Sec22p. Expression of SNARE cysteine derivatives in the same strain produced a cross-linked heterodimer of Bet1p and Sec22p under oxidizing conditions. Moreover, this Bet1p-Sec22p heterodimer formed during in vitro transport reactions when ER-derived vesicles containing the Bet1p derivative fused with Golgi membranes containing the Sec22p derivative. Using this disulfide cross-linking assay, we show that inhibition of transport with anti-Sly1p antibodies blocked formation of the Bet1p-Sec22p heterodimer. In contrast, chelation of divalent cations did not inhibit formation of the Bet1p-Sec22p heterodimer during in vitro transport but potently inhibited Golgi-specific carbohydrate modification of glyco-pro-alpha factor. This data suggests that Ca(2+) is not directly required for membrane fusion between ER-derived vesicles and Golgi-acceptor membranes.

Amino Acid Sequence↗

PubMed2005

ER-Golgi transport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit, Sec24p.

Selective cargo capture into ER-derived vesicles is driven by the Sec24p subunit of the COPII coat, which contains at least three independent cargo-binding sites. One of these, the "A-site," interacts with a NPF motif found on the SNARE, Sed5p. We have characterized the Sec24p-Sed5p interaction through mutation of the putative ER export motifs of Sed5p and the cargo-binding A-site of Sec24p. Mutational analysis of Sed5p suggests that the NPF motif is the dominant ER export signal. Mutation of the NPF binding pocket on Sec24p led to a dramatic reduction in the capture of Sed5p into COPII vesicles, whereas packaging of other ER-Golgi SNAREs was normal. Of all the cargoes tested, only Sed5p was depleted in vesicles made with Sec24p A-site mutants. Surprisingly, vesicles generated with the mutant Sec24p were unable to fuse with the Golgi apparatus. This inability to fuse was not the result of the lack of Sed5p, because vesicles specifically depleted of Sed5p generated by antibody inhibition targeted and fused normally. We propose that the A-site of Sec24p is a multipurpose cargo-binding site that must recognize additional unidentified cargo proteins, at least one of which is essential at a late stage of vesicle fusion.

Biological Transport↗

PubMed2005

A Rab requirement is not bypassed in SLY1-20 suppression.

The Rab GTPase Ypt1p and the large homodimer Uso1p are both required for tethering endoplasmic reticulum-derived vesicles to early Golgi compartments in yeast. Loss-of-function ypt1 and uso1 mutations are suppressed by SLY1-20, a dominant allele that encodes the Sed5p-associated protein, Sly1p. Here, we investigate the mechanism of SLY1-20 suppression. In wild-type strains, Ypt1p can be coimmunoprecipitated with Uso1p; however, in a ypt1Delta/SLY1-20 strain, which lacks this complex, membrane binding of Uso1p was reduced. In spite of Ypt1p depletion, Uso1p-dependent vesicle tethering was not bypassed under the ypt1Delta/SLY1-20 condition. Moreover, tethering and fusion assays with ypt1Delta/SLY1-20 membranes remained sensitive to Rab GDP dissociation inhibitor. These results indicate that an alternative Rab protein satisfies the Ypt1p requirement in Uso1p-dependent tethering when SLY1-20 is expressed. Further genetic and biochemical tests revealed that a related Rab protein, Ypt6, might substitute for Ypt1p in ypt1Delta/SLY1-20 cells. Additional experimentation to address the mechanism of SLY1-20 suppression in a cog2Delta [sec35Delta] strain indicated that the Cog2p subunit of the conserved oligomeric Golgi complex is either functionally redundant or is not directly required for anterograde transport to the Golgi complex.

Carrier Proteins↗

PubMed2005

Yos1p is a novel subunit of the Yip1p-Yif1p complex and is required for transport between the endoplasmic reticulum and the Golgi complex.

Yeast Yip1p is a member of a conserved family of transmembrane proteins that interact with Rab GTPases. Previous studies also have indicated a role for Yip1p in the biogenesis of endoplasmic reticulum (ER)-derived COPII transport vesicles. In this report, we describe the identification and characterization of the uncharacterized open reading frame YER074W-A as a novel multicopy suppressor of the thermosensitive yip1-4 strain. We have termed this gene Yip One Suppressor 1 (YOS1). Yos1p is essential for growth and for function of the secretory pathway; depletion or inactivation of Yos1p blocks transport between the ER and the Golgi complex. YOS1 encodes an integral membrane protein of 87 amino acids that is conserved in eukaryotes. Yos1p localizes to ER and Golgi membranes and is efficiently packaged into ER-derived COPII transport vesicles. Yos1p associates with Yip1p and Yif1p, indicating Yos1p is a novel subunit of the Yip1p-Yif1p complex.

Adaptor Proteins, Vesicular Transport↗

PubMed2004

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.

COP-Coated Vesicles↗

PubMed2004

Proteomics of endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membranes from brefeldin A-treated HepG2 cells identifies ERGIC-32, a new cycling protein that interacts with human Erv46.

Cycling proteins play important roles in the organization and function of the early secretory pathway by participating in membrane traffic and selective transport of cargo between the endoplasmic reticulum (ER), the intermediate compartment (ERGIC), and the Golgi. To identify new cycling proteins, we have developed a novel procedure for the purification of ERGIC membranes from HepG2 cells treated with brefeldin A, a drug known to accumulate cycling proteins in the ERGIC. Membranes enriched 110-fold over the homogenate for ERGIC-53 were obtained and analyzed by mass spectrometry. Major proteins corresponded to established and putative cargo receptors and components mediating protein maturation and membrane traffic. Among the uncharacterized proteins, a 32-kDa protein termed ERGIC-32 is a novel cycling membrane protein with sequence homology to Erv41p and Erv46p, two proteins enriched in COPII vesicles of yeast. ERGIC-32 localizes to the ERGIC and partially colocalizes with the human homologs of Erv41p and Erv46p, which mainly localize to the cis-Golgi. ERGIC-32 interacts with human Erv46 (hErv46) as revealed by covalent cross-linking and mistargeting experiments, and silencing of ERGIC-32 by small interfering RNAs increases the turnover of hErv46. We propose that ERGIC-32 functions as a modulator of the hErv41-hErv46 complex by stabilizing hErv46. Our novel approach for the isolation of the ERGIC from BFA-treated cells may ultimately lead to the identification of all proteins rapidly cycling early in the secretory pathway.

Amino Acid Sequence↗

PubMed2004

Sec22p export from the endoplasmic reticulum is independent of SNARE pairing.

Molecularly distinct sets of SNARE proteins localize to specific intracellular compartments and catalyze membrane fusion events. Although their central role in membrane fusion is appreciated, little is known about the mechanisms by which individual SNARE proteins are targeted to specific organelles. Here we investigated functional domains in Sec22p that direct this SNARE protein to the endoplasmic reticulum (ER), to Golgi membranes, and into SNARE complexes with Bet1p, Bos1p, and Sed5p. A series of Sec22p deletion mutants were monitored in COPII budding assays, subcellular fractionation gradients, and SNARE complex immunoprecipitations. We found that the N-terminal "profilin-like" domain of Sec22p was required but not sufficient for COPII-dependent export of Sec22p from the ER. Interestingly, versions of Sec22p that lacked the N-terminal domain were assembled into ER/Golgi SNARE complexes. Analyses of Sec22p SNARE domain mutants revealed a second signal within the SNARE motif (between layers -4 and -1) that was required for efficient ER export. Other SNARE domain mutants that contained this signal were efficiently packaged into COPII vesicles but failed to assemble into SNARE complexes. Together these results indicated that SNARE complex formation is neither required nor sufficient for Sec22p packaging into COPII transport vesicles and subsequent targeting to the Golgi complex. We propose that the COPII budding machinery has a preference for unassembled ER/Golgi SNARE proteins.

COP-Coated Vesicles↗

PubMed2004

Genetic analysis of yeast Yip1p function reveals a requirement for Golgi-localized rab proteins and rab-Guanine nucleotide dissociation inhibitor.

Yip1p is the first identified Rab-interacting membrane protein and the founder member of the YIP1 family, with both orthologs and paralogs found in all eukaryotic genomes. The exact role of Yip1p is unclear; YIP1 is an essential gene and defective alleles severely disrupt membrane transport and inhibit ER vesicle budding. Yip1p has the ability to physically interact with Rab proteins and the nature of this interaction has led to suggestions that Yip1p may function in the process by which Rab proteins translocate between cytosol and membranes. In this study we have investigated the physiological requirements for Yip1p action. Yip1p function requires Rab-GDI and Rab proteins, and several mutations that abrogate Yip1p function lack Rab-interacting capability. We have previously shown that Yip1p in detergent extracts has the capability to physically interact with Rab proteins in a promiscuous manner; however, a genetic analysis that covers every yeast Rab reveals that the Rab requirement in vivo is exclusively confined to a subset of Rab proteins that are localized to the Golgi apparatus.

COP-Coated Vesicles↗

PubMed2003

A role for Yip1p in COPII vesicle biogenesis.

Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates. Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding. Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation. We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis.

COP-Coated Vesicles↗

PubMed2003

Molecular recognition of cargo by the COPII complex: a most accommodating coat.

The molecular mechanism by which diverse cargo proteins are recognized and exported from the ER has been unclear. Two papers in this issue of Cell add clarity by mapping multiple cargo recognition sites in the Sec24 subunit of the COPII coat complex and demonstrating roles for these sites in export of specific protein cargos from the ER.

Binding Sites↗

PubMed2003

Mammalian Erv46 localizes to the endoplasmic reticulum-Golgi intermediate compartment and to cis-Golgi cisternae.

Yeast endoplasmic reticulum (ER) vesicle protein Erv46p is a novel membrane protein involved in transport through the early secretory pathway. Investigation of mammalian Erv46 (mErv46) reveals that it is broadly expressed in tissues and protein-secreting cells. By immunofluorescence microscopy, mErv46 displays a crescent-shaped perinuclear staining pattern that is characteristic of the Golgi complex. Quantitative immunoelectron microscopy indicates that mErv46 is restricted to the cis face of the Golgi apparatus and to vesicular tubular structures between the transitional ER and cis-Golgi. Minor amounts of mErv46 reside in ER membranes and later Golgi cisternae. On Brefeldin A treatment, mErv46 redistributes to punctate structures that costain for ERGIC53. Depletion of mErv46 protein by RNA interference caused no apparent structural changes in the intermediate compartment or Golgi complex. These findings place mErv46 in a group of itinerant proteins that cycle between the ER and Golgi compartments such as ERGIC53 and the p24 proteins.

Animals↗

PubMed2003

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.

Animals↗

PubMed2002

The Erv41p-Erv46p complex: multiple export signals are required in trans for COPII-dependent transport from the ER.

Erv41p and Erv46p form an integral membrane protein complex that cycles between the endoplasmic reticulum (ER) and Golgi. Both proteins contain a large lumenal domain and short N- and C-terminal tail sequences exposed to the cytosol. The coat protein complex II (COPII) packages the Erv41p-Erv46p complex into ER-derived vesicles for delivery to the Golgi. We determined signals in the Erv41p-Erv46p complex that are required for COPII-dependent export from the ER. Mutants lacking the Erv41p or Erv46p C-terminus accumulated in the ER and were not packaged efficiently into vesicles. We identified an isoleucine-leucine sequence in the Erv41p tail that was required for COPII binding and inclusion of the complex into vesicles. This signal was sufficient for COPII binding but not for ER export. The Erv46p tail contains a phenylalanine-tyrosine sequence required together with the isoleucine-leucine signal in Erv41p for export of the complex. Surprisingly, Erv41p- Erv46p tail-swapped chimeras were not exported from the ER, indicating that signals in both the Erv41p and the Erv46p tail sequences are required in a specific orientation for efficient packaging of the Erv41p-Erv46p complex.

Amino Acid Motifs↗

PubMed2002

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.

Animals↗

PubMed2002

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.

COP-Coated Vesicles↗

PubMed2002

Erv14p directs a transmembrane secretory protein into COPII-coated transport vesicles.

Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast. Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface. Herein, we show that Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles. Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p. When mutations are introduced into this loop, an Erv14p-Axl2p complex accumulates in the endoplasmic reticulum, suggesting that Erv14p links Axl2p to the COPII coat. Based on these results and further genetic experiments, we propose Erv14p coordinates COPII vesicle formation with incorporation of specific secretory cargo.

Amino Acid Sequence↗