Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae

The Kex2 protein of the yeast Saccharomyces cerevisiae is a membrane-bound, Ca2(+)-dependent serine protease that cleaves the precursors of the mating pheromone alpha-factor and the M1 killer toxin at pairs of basic residues during their transport through the secretory pathway. To begin to characterize the intracellular locus of Kex2-dependent proteolytic processing, we have examined the subcellular distribution of Kex2 protein in yeast by indirect immunofluorescence. Kex2 protein is located at multiple, discrete sites within wild-type yeast cells (average, 3.0 +/- 1.7/mother cell). Qualitatively similar fluorescence patterns are observed at elevated levels of expression, but no signal is found in cells lacking the KEX2 gene. Structures containing Kex2 protein are not concentrated at a perinuclear location, but are distributed throughout the cytoplasm at all phases of the cell cycle. Kex2-containing structures appear in the bud at an early, premitotic stage. Analysis of conditional secretory (sec) mutants demonstrates that Kex2 protein ordinarily progresses from the ER to the Golgi but is not incorporated into secretory vesicles, consistent with the proposed localization of Kex2 protein to the yeast Golgi complex.     

ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway

Resident proteins of the ER lumen carry a specific tetrapeptide signal (KDEL or HDEL) that prevents their secretion. We have previously described the isolation of yeast mutants that fail to retain such resident proteins within the cell. Here we describe ERD2, a gene required for retention. It encodes a 26 kd integral membrane protein whose abundance determines the efficiency and capacity of the retention system. Reduced expression of ERD2 leads to secretion of proteins bearing the HDEL signal, whereas overexpression of ERD2 improves retention both in wild-type cells and in other mutants. These results are consistent with other evidence that ERD2 encodes the HDEL receptor (see accompanying paper). The gene is also required, perhaps indirectly, for normal protein transport through the Golgi, and hence for growth. We discuss possible roles for ERD2 in the secretory pathway.     

The Saccharomyces cerevisiae SEC20 gene encodes a membrane glycoprotein which is sorted by the HDEL retrieval system.

The SEC20 gene product (Sec20p) is required for endoplasmic reticulum (ER) to Golgi transport in the yeast secretory pathway. We have cloned the SEC20 gene by complementation of the temperature sensitive phenotype of a sec20-1 strain. The DNA sequence predicts a 44 kDa protein with a single membrane-spanning region; Sec20p has an apparent molecular weight of 50 kDa and behaves as an integral membrane protein with carbohydrate modifications that appear to be O-linked. A striking feature of this protein is its C-terminal sequence, which consists of the tetrapeptide HDEL. This signal is known to be required for the retrieval of soluble ER proteins from early Golgi compartments, but has not previously been observed on a membrane protein. The HDEL sequence of Sec20p is not essential for viability but helps to maintain intracellular levels of the protein. Depletion of Sec20p from cells results in the accumulation of an extensive network of ER and clusters of small vesicles. We suggest a possible role for the SEC20 product in the targeting of transport vesicles to the Golgi apparatus.     

Genetic analysis of clathrin function in yeast

The use of yeast mutants to study the function and dynamics of clathrin-coated membranes has offered new insights into clathrin's role in the secretory pathway and has raised additional questions. Most strains of yeast can incur a disruption of clathrin heavy or light chain genes and remain viable. However, in rare cases, alleles of genes other than clathrin affect the viability of clathrin-deficient cells. The relationship of the products of these genes to clathrin awaits clarification. Phenotypic characterization of clathrin-deficient yeast mutants suggests that clathrin is not essential for the generation of secretory pathway transport vesicles at the ER or the Golgi complex but is required for the intracellular retention of a Golgi membrane protein, Kex2p. With this genetic evidence for clathrin's function in vivo, biochemical and genetic experiments can be designed to address the mechanism by which clathrin effects retention of Kex2p. Clathrin-deficient yeast carry out protein secretion, receptor-mediated endocytosis of mating pheromone, and efficient targeting of newly synthesized vacuolar proteins. These observations challenge aspects of clathrin's proposed involvement in protein transport through the secretory pathway and to lysosomes in mammalian cells. However, the differences are beginning to recede in the face of additional experiments; the formation of clathrin coated vesicles is no longer commonly thought to be obligately coupled to transport through the secretory pathway in mammalian cells (Rothman 1986; Brodsky, 1988), and the role of clathrin in retaining a Golgi membrane protein in yeast may have its precedents in receptor-mediated endocytosis by mammalian cells or in secretory granule formation in endocrine cells. A unified theory of clathrin function is emerging (Brodsky, 1988) which suggests that the clathrin coat assemblage (clathrin heavy and light chains and the associated proteins) acts as a facilitator of intracellular protein transport by sorting and concentrating cargo molecules. The results from studies of clathrin-deficient yeast support this theory. Future experiments will determine whether clathrin provides its functions at different transport stages in different organisms or whether all eukaryotic cells employ clathrin at the same stages of intracellular protein transport.     

Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complex

An insulin-containing fusion protein (ICFP, encoding the yeast prepro-alpha factor leader peptide fused via a lysine-arginine cleavage site to a single chain insulin) has been expressed in Saccharomyces cerevisiae where it is inefficiently secreted. Single gene disruptions have been identified that cause enhanced immunoreactive insulin secretion (eis). Five out of six eis mutants prove to be vacuolar protein sorting (vps)8, vps35, vps13, vps4, and vps36, which affect Golgi<-->endosome trafficking. Indeed, in wild-type yeast insulin is ultimately delivered to the vacuole, whereas vps mutants secrete primarily unprocessed ICFP. Disruption of KEX2, which blocks intracellular processing to insulin, quantitatively reroutes ICFP to the cell surface, whereas loss of the Vps10p sorting receptor is without effect. Secretion of unprocessed ICFP is not based on a dominant secretion signal in the alpha-leader peptide. Although insulin sorting mediated by Kex2p is saturable, Kex2p functions not as a sorting receptor but as a protease: replacement of Kex2p by truncated secretory Kex2p (which travels from Golgi to cell surface) still causes endoproteolytic processing and intracellular insulin retention. Endoproteolysis promotes a change in insulin's biophysical properties. B5His residues normally participate in multimeric insulin packing; a point mutation at this position permits ICFP processing but causes the majority of processed insulin to be secreted. The data argue that multimeric assembly consequent to endoproteolytic maturation regulates insulin sorting in the secretory pathway.     

Reconstitution of Retrograde Transport from the Golgi to the ER In Vitro

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone α-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369–377). Retrieval depends on the HDEL sequence; the α-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.     

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.     

The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p.

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-terminal cytosolic tail. Mutation of the TLS accelerates transport of Kex2p to the vacuole by an intracellular (SEC1-independent) pathway. In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed by examining the effects of a temperature-sensitive CHC1 allele on trafficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TLS mutant forms of Kex2p to behave identically. All forms of Kex2p appeared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasing to that of wild-type Kex2p. After inactivation of clathrin heavy chain, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fusion to the plasma membrane, indicating that transport to the cell surface was required for degradation by vacuolar proteolysis. Finally, after clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both chc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localization are independent of the Kex2p C-terminal cytosolic tail. Although these results neither prove nor rule out a direct interaction between the Kex2 TLS and a clathrin-dependent structure, they do imply that clathrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.     

A novel Kex2 enzyme can process the proregion of the yeast alpha-factor leader in the endoplasmic reticulum instead of in the Golgi.

The prepro sequence of the yeast prepro-alpha-factor, usually referred to as the alpha-factor leader, has often been used for the efficient secretion of heterologous proteins from the yeast Saccharomyces cerevisiae. The alpha-factor leader consists of a 19-amino acid N-terminal pre or signal sequence followed by a 66-amino acid proregion. After removal of the signal sequence during membrane translocation, the proregion is cleaved from the precursor protein by the Kex2 endoprotease only in a late Golgi compartment. Here we report that a modified Kex2 enzyme, containing at the C-terminus the HDEL tetrapeptide, cleaves the proregion from the alpha-factor leader--human insulin like growth factor-1 fusion protein in the endoplasmic reticulum. The processing of pro-proteins earlier in the secretion pathway could be helpful in defining the cellular function of the proregions present naturally in various eucaryotic precursor proteins.     

Requirement for neo1p in retrograde transport from the Golgi complex to the endoplasmic reticulum

Neo1p from Saccharomyces cerevisiae is an essential P-type ATPase and potential aminophospholipid translocase (flippase) in the Drs2p family. We have previously implicated Drs2p in protein transport steps in the late secretory pathway requiring ADP-ribosylation factor (ARF) and clathrin. Here, we present evidence that epitope-tagged Neo1p localizes to the endoplasmic reticulum (ER) and Golgi complex and is required for a retrograde transport pathway between these organelles. Using conditional alleles of NEO1, we find that loss of Neo1p function causes cargo-specific defects in anterograde protein transport early in the secretory pathway and perturbs glycosylation in the Golgi complex. Rer1-GFP, a protein that cycles between the ER and Golgi complex in COPI and COPII vesicles, is mislocalized to the vacuole in neo1-ts at the nonpermissive temperature. These phenotypes suggest that the anterograde protein transport defect is a secondary consequence of a defect in a COPI-dependent retrograde pathway. We propose that loss of lipid asymmetry in the cis Golgi perturbs retrograde protein transport to the ER.     

Mutations in a highly conserved region of the Arf1p activator GEA2 block anterograde Golgi transport but not COPI recruitment to membranes

We have identified an important functional region of the yeast Arf1 activator Gea2p upstream of the catalytic Sec7 domain and characterized a set of temperature-sensitive (ts) mutants with amino acid substitutions in this region. These gea2-ts mutants block or slow transport of proteins traversing the secretory pathway at exit from the endoplasmic reticulum (ER) and the early Golgi, and accumulate both ER and early Golgi membranes. No defects in two types of retrograde trafficking/sorting assays were observed. We find that a substantial amount of COPI is associated with Golgi membranes in the gea2-ts mutants, even after prolonged incubation at the nonpermissive temperature. COPI in these mutants is released from Golgi membranes by brefeldin A, a drug that binds directly to Gea2p and blocks Arf1 activation. Our results demonstrate that COPI function in sorting of at least three retrograde cargo proteins within the Golgi is not perturbed in these mutants, but that forward transport is severely inhibited. Hence this region of Gea2p upstream of the Sec7 domain plays a role in anterograde transport that is independent of its role in recruiting COPI for retrograde transport, at least of a subset of Golgi-ER cargo.     

Retrotranslocation of a viral A/B toxin from the yeast endoplasmic reticulum is independent of ubiquitination and ERAD

K28 is a viral A/B toxin that traverses eukaryotic cells by endocytosis and retrograde transport through the secretory pathway. Here we show that toxin retrotranslocation from the endoplasmic reticulum (ER) requires Kar2p/BiP, Pdi1p, Scj1p, Jem1p, and proper maintenance of Ca(2+) homeostasis. Neither cytosolic chaperones nor Cdc48p/Ufd1p/Npl4p complex components or proteasome activity are required for ER exit, indicating that K28 retrotranslocation is mechanistically different from classical ER-associated protein degradation (ERAD). We demonstrate that K28 exits the ER in a heterodimeric but unfolded conformation and dissociates into its subunits as it emerges into the cytosol where beta is ubiquitinated and degraded. ER export and in vivo toxicity were not affected in a lysine-free K28 variant nor under conditions when ubiquitination and proteasome activity was blocked. In contrast, toxin uptake from the plasma membrane required Ubc4p (E2) and Rsp5p (E3) and intoxicated ubc4 and rsp5 mutants accumulate K28 at the cell surface incapable of toxin internalization. We propose a model in which ubiquitination is involved in the endocytic pathway of the toxin, while ER-to-cytosol retrotranslocation is independent of ubiquitination, ERAD and proteasome activity.     

Expression and degradation of the cystic fibrosis transmembrane conductance regulator in Saccharomyces cerevisiae

Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Yeast mutants, defective at various steps of the secretory pathway, have been used to dissect the mechanisms of biosynthetic processing and intracellular transport of several proteins. To exploit these yeast mutants, we have employed an expression system in which the CFTR gene is driven by the promoter of a structurally related yeast ABC protein, Pdr5p. Pulse-chase experiments revealed a turnover rate similar to that of nascent CFTR in mammalian cells. Immunofluorescence microscopy showed that most CFTR colocalized with the endoplasmic reticulum (ER) marker protein Kar2p and not with a vacuolar marker. Degradation was not influenced by the vacuolar protease mutants Pep4p and Prb1p but was sensitive to the proteasome inhibitor lactacystin beta-lactone. Blocking ER-to-Golgi transit with the sec18-1 mutant had little influence on turnover indicating that it occurred primarily in the ER compartment. Degradation was slowed in cells deficient in the ER degradation protein Der3p as well as the ubiquitin-conjugating enzymes Ubc6p and Ubc7p. Finally a mutation (sec61-2) in the translocon protein Sec61p that prevents retrotranslocation across the ER membrane also blocked degradation. These results indicate that whereas approximately 75% of nascent wild-type CFTR is degraded at the ER of mammalian cells virtually all of the protein meets this fate on heterologous expression in Saccharomyces cerevisiae.     

The ERD2 gene determines the specificity of the luminal ER protein retention system

Luminal ER proteins carry a signal at their C terminus that prevents their secretion; in S. cerevisiae this signal is the tetrapeptide HDEL. Indirect evidence suggests that HDEL is recognized by a receptor that retrieves ER proteins from the secretory pathway and returns them to the ER, and a candidate for this receptor is the product of the ERD2 gene (see accompanying paper). We show here that presumptive ER proteins from the budding yeast K. lactis can terminate either with HDEL or, in the case of BiP, with DDEL. S. cerevisiae does not efficiently recognize DDEL as a retention signal, but exchange of its ERD2 gene for the corresponding gene from K. lactis allows equal recognition of DDEL and HDEL. Thus the specificity of the retention system is determined by the ERD2 gene. We conclude that ERD2 encodes the receptor that sorts luminal ER proteins.     

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.     

Kex2-dependent invertase secretion as a tool to study the targeting of transmembrane proteins which are involved in ER-->Golgi transport in yeast.

Mutants were isolated that are defective in the retention of a transmembrane protein in the early secretory compartments in yeast. A series of hybrid proteins was tested for their use in the selection of such mutants. Each of these hybrid proteins consisted of a type II transmembrane protein (Nin/Cout) and invertase (Suc2) as a reporter separated by a peptide linker containing a cleavage site for the Golgi protease Kex2. The integral membrane proteins which were used--Sec12p, Sec22/Sly2p or Bet1/Sly12p--are all known to be required for ER-->Golgi transport in yeast. Invertase was readily cleaved from the fusions containing Sec22/Sly2p or Bet1/Sly12p as the membrane anchoring part. In contrast, Sec12--invertase expressing transformants required mutations in either of two different genes for Kex2-dependent invertase secretion. The mutant showing the stronger retention defect (rer1) was used to clone the corresponding gene. RER1 represents the first reading frame left of the centromere of chromosome III. Cells carrying a disruption of the RER1 gene are viable and show the same mislocalizing phenotype as the original mutants. The Rer1 protein, as deduced from the nucleotide sequence, contains four transmembrane domains. It has been suggested before that Sec12p cycles between the ER and the cis-Golgi compartment. Some results obtained by using Sec12-invertase and the rer1 mutants resemble observations on the retention of Golgi-resident glycosyltransferases and viral proteins in mammalian cells. For instance, retention of Sec12-invertase is non-saturable and the membrane-spanning domain of Sec12p seems to constitute an important targeting signal.     

Yeast Golgi-localized, gamma-Ear-containing, ADP-ribosylation factor-binding proteins are but adaptor protein-1 is not required for cell-free transport of membrane proteins from the trans-Golgi network to the prevacuolar compartment

Golgi-localized, γ-Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN–PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the β subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Δ membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Δ mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome.     

Prohormone processing by yeast proteases.

Investigations of the precursors of alpha-pheromone and killer toxin in the yeast Saccharomyces cerevisiae have defined the genes coding (KEX1 and KEX2) for the proteases which are responsible for their processing. In addition to processing at pairs of basic residues it is evident that yeast can also process at monobasic sites. We present data on the Kex1p and Kex2p enzymes, their cellular localization, and their post-translational modification. In addition initial characterisation of the monobasic specific protease and the isolation of mutants defective in this activity are presented. The use of the yeast system as a model for the processing of mammalian prohormones is discussed.     

A mutant Kex2 enzyme with a C-terminal HDEL sequence releases correctly folded human insulin-like growth factor-1 from a precursor accumulated in the yeast endoplasmic reticulum.

Mutations in the pro region of the yeast DNA hybrid of prepro-alpha-factor and human insulin-like growth factor-1 (IGF-1) cause the accumulation, in the yeast Saccharomyces cerevisiae, of an unglycosylated precursor protein where the pre sequence is missing. The prepro sequence of the prepro-alpha-factor consists of a pre or signal sequence and a proregion which possesses three sites for N-glycosylation. Isolation of a precursor, where the pro region is still linked to IGF-1 through a pair of dibasic amino acid residues, implies that the polypeptide may have translocated into the endoplasmic reticulum (ER) but has not been processed by the Golgi membrane-bound Kex2 endoprotease. However, the lack of any N-glycosylation in the translocated polypeptide is surprising. The mutated pro region, can be processed, in vitro, by treatment with a soluble form of the Kex2 enzyme. It is also possible to release the pro region, in vivo, by coexpressing a mutant Kex2 protease which is partially retained in the ER with the help of the C-terminal tetrapeptide sequence, HDEL. The mature IGF-1, which is secreted from the intracellular pool of precursor proteins, is predominantly an active, monomeric molecule, corroborating observations that early removal of the pro region before folding in the ER helps to prevent aberrant intermolecular disulfide-bond formation in IGF-1. These results have revealed the utility of the ER-retained Kex2 enzyme as a novel in vivo biochemical tool.