A Novel Membrane Protein Complex on the Endoplasmic Reticulum and Early Golgi Compartments in the Yeast Saccharomyces cerevisiae

A novel membrane protein, Yml067c in the systematic ORF name, was discovered as a component of immunoisolated vesicles of the early Golgi compartment of the yeast Saccharomyces cerevisiae (Cho et al., FEBS Lett. 469, 151-154 (2000)). Conserved sequences having sequence similarity to Yml067c were widely distributed in the eukaryotes and one of them, Yal042w, was found in the Saccharomyces genome database. In the yeast cell, Yml067c and Yal042w were found to form a heterooligomeric complex by immunoprecipitation of their tagged derivatives from the detergent-solubilized membrane. Cell fractionation and indirect immunofluorescent staining indicated that the majority of these proteins were localized on the ER membrane. Therfore, the Yml067c-Yal042w complex should shuttle between the ER and the early Golgi compartment as well as the p24-family proteins.     

Proteins in the early golgi compartment of Saccharomyces cerevisiae immunoisolated by Sed5p

The yeast tSNARE Sed5p is considered to mainly reside in the early Golgi compartment at the steady state of its intracellular cycling. To better understand this compartment, we immunoisolated a membrane subfraction having Sed5p on the surface (the Sed5 vesicles). Immunoblot studies showed that considerable portions (20-30%) of the Golgi mannosyltransferases (Mnt1p, Van1p, and Mnn9p) were simultaneously recovered while the late Golgi (Kex2p) or endoplasmic reticulum (Sec71p) proteins were almost excluded. The N-terminal sequences of the polypeptides detectable by Coomassie blue staining indicated that the prominent components of the Sed5 vesicles include Anp1p, Emp24p, Erv25p, Erp1p, Ypt52p, and a putative membrane protein of unknown function (Yml067c).     

Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant

The sec18 and sec23 secretory mutants of Saccharomyces cerevisiae have previously been shown to exhibit temperature-conditional defects in protein transport from the ER to the Golgi complex (Novick, P., S. Ferro, and R. Schekman, 1981. Cell. 25:461-469). We have found that the Sec18 and Sec23 protein functions are rapidly inactivated upon shifting mutant cells to the nonpermissive temperature (less than 1 min). This has permitted an analysis of the potential role these SEC gene products play in transport events distal to the ER. The sec-dependent transport of alpha-factor (alpha f) and carboxypeptidase Y (CPY) biosynthetic intermediates present throughout the secretory pathway was monitored in temperature shift experiments. We found that Sec18p/NSF function was required sequentially for protein transport from the ER to the Golgi complex, through multiple Golgi compartments and from the Golgi complex to the cell surface. In contrast, Sec23p function was required in the Golgi complex, but only for transport of alpha f out of an early compartment. Together, these studies define at least three functionally distinct Golgi compartments in yeast. From cis to trans these compartments contain: (a) An alpha 1----6 mannosyltransferase; (b) an alpha 1----3 mannosyltransferase; and (c) the Kex2 endopeptidase. Surprisingly, we also found that a pool of Golgi-modified CPY (p2 CPY) located in a compartment distal to the alpha 1----3 mannosyltransferase does not require Sec18p function for final delivery to the vacuole. This compartment appears to be equivalent to the Kex2 compartment as we show that a novel vacuolar CPY-alpha f-invertase fusion protein undergoes efficient Kex2-dependent cleavage resulting in the secretion of invertase. We propose that this Kex2 compartment is the site in which vacuolar proteins are sorted from proteins destined to be secreted.     

Brefeldin A reversibly blocks early but not late protein transport steps in the yeast secretory pathway.

We have found that brefeldin A (BFA) inhibited the growth of an ise1 mutant of Saccharomyces cerevisiae. Genetic complementation and mapping studies demonstrated that ise1 was allelic to erg6, a gene required for the biosynthesis of the principal membrane sterol of yeast, ergosterol. Treatment of ise1 cells with BFA resulted in an immediate block in protein transport through the secretory pathway. Vacuolar carboxypeptidase Y (CPY) and the secreted pheromone alpha-factor accumulated as both the core glycosylated (ER) and alpha 1,6 mannosylated (early Golgi) forms in drug-treated cells. The modification of alpha-factor with alpha 1,6 mannose in BFA-treated cells did not appear to result from retrograde transport of the alpha 1,6 mannosyl-transferase into the ER. We found that transport of CPY from medial and late Golgi compartments to the vacuole was unaffected by BFA, nor was secretion of alpha 1,3 mannosylated alpha-factor or invertase blocked by BFA. The effects of BFA on the secretory pathway were also reversible after brief exposure (< 40 min) to the drug. We suggest that the primary effect of BFA in S. cerevisiae is restricted to the ER and the alpha 1,6 mannosyltransferase compartment of the Golgi complex.     

Direct interaction of the Golgi membrane with the endoplasmic reticulum membrane caused by nordihydroguaiaretic acid

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, blocks protein transport from the endoplasmic reticulum (ER) to the Golgi complex and induces the redistribution of Golgi proteins into the ER. We investigated characteristics of NDGA-induced retrograde movement of the Golgi proteins to the ER. At an early stage of incubation of cells with NDGA, the Golgi complex formed convoluted membrane aggregates. Electron microscopy revealed that these aggregates directly interact en bloc with the ER membrane. The direct interaction and subsequent incorporation of the Golgi proteins into the ER were found to be temperature-dependent. The protein of ER-Golgi intermediate compartment (ERGIC), ERGIC53, was rapidly accumulated in the Golgi upon treatment with NDGA. This accumulation was significantly inhibited by low temperature at 15 degrees C. Under the condition, the redistribution of the Golgi proteins into the ER as well as the direct interaction between the ER and the Golgi by NDGA were also inhibited, suggesting an important role of the ERGIC in the retrograde movement. In contrast, the low temperature did not inhibit formation of the Golgi aggregates by NDGA. Taken together, these results suggest that NDGA causes the redistribution of the Golgi proteins into the ER through the direct connections between the Golgi, the ERGIC, and the ER.     

Vps52p, Vps53p, and Vps54p form a novel multisubunit complex required for protein sorting at the yeast late Golgi

The late Golgi of the yeast Saccharomyces cerevisiae receives membrane traffic from the secretory pathway as well as retrograde traffic from post-Golgi compartments, but the machinery that regulates these vesicle-docking and fusion events has not been characterized. We have identified three components of a novel protein complex that is required for protein sorting at the yeast late Golgi compartment. Mutation of VPS52, VPS53, or VPS54 results in the missorting of 70% of the vacuolar hydrolase carboxypeptidase Y as well as the mislocalization of late Golgi membrane proteins to the vacuole, whereas protein traffic through the early part of the Golgi complex is unaffected. A vps52/53/54 triple mutant strain is phenotypically indistinguishable from each of the single mutants, consistent with the model that all three are required for a common step in membrane transport. Native coimmunoprecipitation experiments indicate that Vps52p, Vps53p, and Vps54p are associated in a 1:1:1 complex that sediments as a single peak on sucrose velocity gradients. This complex, which exists both in a soluble pool and as a peripheral component of a membrane fraction, colocalizes with markers of the yeast late Golgi by immunofluorescence microscopy. Together, the phenotypic and biochemical data suggest that VPS52, VPS53, and VPS54 are required for the retrograde transport of Golgi membrane proteins from an endosomal/prevacuolar compartment. The Vps52/53/54 complex joins a growing list of distinct multisubunit complexes that regulate membrane-trafficking events.     

Recycling of proteins from the Golgi compartment to the ER in yeast

In the yeast Saccharomyces cerevisiae, the carboxyl terminal sequence His-Asp-Glu-Leu (HDEL) has been shown to function as an ER retention sequence (Pelham, H. R. B., K. G. Hardwick, and M. J. Lewis. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1757-1762). To examine the mechanism of retention of soluble ER proteins in yeast, we have analyzed the expression of a preproalpha factor fusion protein, tagged at the carboxyl terminus with the HDEL sequence. We demonstrate that this fusion protein, expressed in vivo, accumulates intracellularly as a precursor containing both ER and Golgi-specific oligosaccharide modifications. The Golgi-specific carbohydrate modification, which occurs in a SEC18-dependent manner, consists of alpha 1-6 mannose linkages, with no detectable alpha 1-3 mannose additions, indicating that the transit of the HDEL-tagged fusion protein is confined to an early Golgi compartment. Results obtained from the fractionation of subcellular organelles from yeast expressing HDEL-tagged fusion proteins suggest that the Golgi-modified species are present in the ER. Overexpression of HDEL-tagged preproalpha factor results in the secretion of an endogenous HDEL-containing protein, demonstrating that the HDEL recognition system can be saturated. These results support the model in which the retention of these proteins in the ER is dependent on their receptor-mediated recycling from the Golgi complex back to the ER.     

The functioning of the yeast Golgi apparatus requires an ER protein encoded by ANP1, a member of a new family of genes affecting the secretory pathway.

Mnt1p is an alpha 1.2-mannosyltransferase which resides in an early compartment of the Saccharomyces cerevisiae Golgi apparatus. We have shown that the signal-anchor region is sufficient, and the transmembrane domain necessary, for its normal Golgi localization. This is similar to the transmembrane domain-mediated retention of mammalian glycosyltransferases, and distinct from the tail-mediated recycling retention of certain mammalian and yeast trans-Golgi proteins. To examine the mechanism involved in transmembrane domain-mediated retention, we have isolated six classes of mutants which fail to retain Mnt1p-reporter fusions in the early Golgi. These mutants all show additional phenotypes which are consistent with alterations in Golgi function. We have called the mutant classes 'gem', for Golgi enzyme maintenance. GEM3 is identical to the previously cloned gene ANP1, and homologous to VAN1 and MNN9. Together, these define a new class of proteins involved in the organization and functioning of the secretory pathway. Interestingly, Anp1p is localized to the endoplasmic reticulum (ER), implying that some function of the ER is required to maintain a functional Golgi apparatus.     

Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast

The Saccharomyces cerevisiae Wbp1 protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysine (KKXX) motif. This motif has previously been shown to direct Golgi-to-ER retrieval of type I membrane proteins in mammalian cells (Jackson, M. R., T. Nilsson, and P. A. Peterson. 1993. J. Cell Biol. 121: 317-333). To analyze the role of this motif in yeast, we constructed a SUC2-WBP1 chimera consisting of the coding sequence for the normally secreted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytoplasmic tail) of Wbplp. Carbohydrate analysis of the invertase-Wbp1 fusion protein using mannose linkage-specific antiserum demonstrated that the fusion protein was efficiently modified by the early Golgi initial alpha 1,6 mannosyltransferase (Och1p). Subcellular fractionation revealed that > 90% of the alpha 1,6 mannose-modified fusion protein colocalized with the ER (Wbp1p) and not with the Golgi Och1p-containing compartment or other membrane fractions. Amino acid changes within the dily sine motif (KK-->QK, KQ, or QQ) did not change the kinetics of initial alpha 1,6 mannose modification of the fusion protein but did dramatically increase the rate of modification by more distal Golgi (elongating alpha 1,6 and alpha 1,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-dependent manner. While amino acids surrounding the dilysine motif played only a minor role in retention ability, mutations that altered the position of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our results indicate that the KKXX motif does not simply retain proteins in the ER but rather directs their rapid retrieval from a novel, Och1p-containing early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate COOH-terminal position which represents the most important features of this sorting determinant.     

Immunoisolaton of the yeast Golgi subcompartments and characterization of a novel membrane protein, Svp26, discovered in the Sed5-containing compartments

The Golgi apparatus consists of a set of vesicular compartments which are distinguished by their marker proteins. These compartments are physically separated in the Saccharomyces cerevisiae cell. To characterize them extensively, we immunoisolated vesicles carrying either of the SNAREs Sed5 or Tlg2, the markers of the early and late Golgi compartments, respectively, and analyzed the membrane proteins. The composition of proteins was mostly consistent with the position of each compartment in the traffic. We found six uncharacterized but evolutionarily conserved proteins and named them Svp26 (Sed5 compartment vesicle protein of 26 kDa), Tvp38, Tvp23, Tvp18, Tvp15 (Tlg2 compartment vesicle proteins of 38, 23, 18, and 15 kDa), and Gvp36 (Golgi vesicle protein of 36 kDa). The localization of Svp26 in the early Golgi compartment was confirmed by microscopic and biochemical means. Immunoprecipitation indicated that Svp26 binds to itself and a Golgi mannosyltransferase, Ktr3. In the absence of Svp26, a considerable portion of Ktr3 was mislocalized in the endoplasmic reticulum. Our data suggest that Svp26 has a novel role in retention of a subset of membrane proteins in the early Golgi compartments.     

The p24 family member p23 is required for early embryonic development

The p24 family of type I integral-membrane proteins, which are localised in the endoplasmic reticulum (ER), the intermediate compartment and the Golgi apparatus, are thought to function as receptors for cargo exit from the ER and in transport vesicle formation. Members of the p24 family have been found in a molecular complex and are enriched in COPI-coated vesicles, which are involved in membrane traffic between the ER and Golgi complex. Although expressed abundantly, simultaneous deletion of several family members does not appear to affect cell viability and protein secretion in yeast. In order to gain more insights into the physiological roles of different p24 proteins, we generated mice deficient in the expression of one family member, p23 (also called 24delta1, see for alternative nomenclature). In contrast to yeast genetics, in mice disruption of both p23 alleles resulted in early embryonic lethality. Inactivation of one allele led not only to reduced levels of p23 itself but also to reduced levels of other family members. The reduction in steady-state protein levels also induced structural changes in the Golgi apparatus, such as the formation of dilated saccules. The generation of mice deficient in p23 expression has revealed an essential and non-redundant role for p23 in the earliest stages of mammalian development. It has also provided genetic evidence for the participation of p24 family members in oligomeric complexes and indicates a structural role for these proteins in maintaining the integrity of the early secretory pathway.     

Systems cell biology

Upon starvation, Grh1, a peripheral membrane protein located at endoplasmic reticulum (ER) exit sites and early Golgi in Saccharomyces cerevisiae under growth conditions, relocates to a compartment called compartment for unconventional protein secretion (CUPS). Here we report that CUPS lack Golgi enzymes, but contain the coat protein complex II (COPII) vesicle tethering protein Uso1 and the Golgi t-SNARE Sed5. Interestingly, CUPS biogenesis is independent of COPII- and COPI-mediated membrane transport. Pik1- and Sec7-mediated membrane export from the late Golgi is required for complete assembly of CUPS, and Vps34 is needed for their maintenance. CUPS formation is triggered by glucose, but not nitrogen starvation. Moreover, upon return to growth conditions, CUPS are absorbed into the ER, and not the vacuole. Altogether our findings indicate that CUPS are not specialized autophagosomes as suggested previously. We suggest that starvation triggers relocation of secretory and endosomal membranes, but not their enzymes, to generate CUPS to sort and secrete proteins that do not enter, or are not processed by enzymes of the ER–Golgi pathway of secretion.     

Dynamic retention of TGN membrane proteins in Saccharomyces cerevisiae

In a secretory pathway organelle like the Golgi complex, resident proteins are retained in the face of substantial protein flux to subsequent destinations. Recently, molecular genetic strategies have been used to study membrane protein retention in a compartment of the yeast Saccharomyces cerevisiae that is analogous to the trans Golgi network (TGN) of mammalian cells. These studies have defined retention signals containing aromatic amino acids in the TGN proteins' cytoplasmic domains. The identification of mutants that fail to retain TGN proteins has offered the first glimpse into the components involved in retention. The phenotypes of these mutants suggest that retention involves retrieval of TGN proteins from an endosomal compartment.     

Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway

The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues. Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology. Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked mannose in the ER. The earliest detectable species, I1 (approximately 129 kD), undergoes rapid amino-terminal proteolytic removal of a approximately 9-kD pro-segment yielding species I2 (approximately 120 kD) before arrival at the Golgi complex. Transport to the Golgi complex is marked by extensive elaboration of Ser/Thr-linked chains and minor modification of Asn-linked oligosaccharide. During the latter phase of its lifetime, Kex2 protein undergoes a gradual increase in apparent molecular weight. This final modification serves as a marker for association of Kex2 protease with a late compartment of the yeast Golgi complex in which it is concentrated about 27-fold relative to other secretory proteins.     

Structure and Assembly of the Yeast V-ATPase

The yeast V-ATPase belongs to a family of V-type ATPases present in all eucaryotic organisms. In Saccharomyces cerevisiae the V-ATPase is localized to the membrane of the vacuole as well as the Golgi complex and endosomes. The V-ATPase brings about the acidification of these organelles by the transport of protons coupled to the hydrolysis of ATP. In yeast, the V-ATPase is composed of 13 subunits consisting of a catalytic V₁ domain of peripherally associated proteins and a proton-translocating V₀ domain of integral membrane proteins. The regulatory subunit, Vma13p, was the first V-ATPase subunit to have its crystal structure determined. In addition to proteins forming the functional V-ATPase complex, three ER-localized proteins facilitate the assembly of the V₀ subunits following their translation and insertion into the membrane of the ER. Homologues of the Vma21p assembly factor have been identified in many higher eukaryotes supporting a ubiquitous assembly pathway for this important enzyme complex.     

Retrograde Transport of Golgi-localized Proteins to the ER

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.     

Reticulon 3 is involved in membrane trafficking between the endoplasmic reticulum and Golgi

Reticulons (RTNs) constitute a family of endoplasmic reticulum (ER)-associated proteins with a reticular distribution. Despite the implication of their neuronal isoforms in axonal regeneration, the function of their widely expressed isoforms is largely unknown. In this study, we examined the role of the ubiquitously expressed RTN3 in membrane trafficking. Ectopically expressed RTN3 exhibited heterogeneous patterns; filamentous, reticular, and granular distributions. The ER morphology changed accordingly. In cells where RTN3 displayed a filamentous/reticular distribution, protein transport between the ER and Golgi was blocked, and Golgi proteins were dispersed. In contrast, ERGIC-53, a marker for the ER-Golgi intermediate compartment, accumulated at the perinuclear region, and remained there even after cells were treated with agents that induce redistribution of Golgi proteins to the ER, indicating an inhibition of Golgi-to-ER transport of ERGIC-53. These results suggest that RTN3 plays a role in membrane trafficking in the early secretory pathway.     

The Florey Lecture, 1992. The secretion of proteins by cells.

In eukaryotic cells, protein secretion provides a complex organizational problem. Secretory proteins are first transported, in an unfolded state, across the membrane of the endoplasmic reticulum (ER), and are then carried in small vesicles to the Golgi apparatus and finally to the cell membrane. The ER contains soluble proteins which catalyse the folding of newly synthesized polypeptides. These proteins are sorted from secretory proteins in the Golgi complex: they carry a sorting signal (the tetrapeptide KDEL or a related sequence) that allows them to be selectively retrieved and returned to the ER. This retrieval process also appears to be used by some bacterial toxins to aid their invasion of the cell: these toxins contain KDEL-like sequences and may, in effect, follow the secretory pathway in reverse. The membrane-bound receptor responsible for sorting luminal ER proteins has been identified in yeast by genetic means, and related receptors are found in mammalian cells. Unexpectedly, this receptor has a second role: in yeast it is required to maintain the normal size and function of the Golgi apparatus. By helping to maintain the composition of both ER and Golgi compartments, the KDEL receptor has an important role in the organization of the secretory pathway.