Dual Roles for Ste24p in Yeast a-Factor Maturation: NH2-terminal Proteolysis and COOH-terminal CAAX Processing

Maturation of the Saccharomyces cerevisiae a-factor precursor involves COOH-terminal CAAX processing (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) followed by cleavage of an NH₂-terminal extension (two sequential proteolytic processing steps). The aim of this study is to clarify the precise role of Ste24p, a membrane-spanning zinc metalloprotease, in the proteolytic processing of the a-factor precursor. We demonstrated previously that Ste24p is necessary for the first NH₂-terminal processing step by analysis of radiolabeled a-factor intermediates in vivo (Fujimura-Kamada, K., F.J. Nouvet, and S. Michaelis. 1997. J. Cell Biol. 136:271–285). In contrast, using an in vitro protease assay, others showed that Ste24p (Afc1p) and another gene product, Rce1p, share partial overlapping function as COOH-terminal CAAX proteases (Boyartchuk, V.L., M.N. Ashby, and J. Rine. 1997. Science. 275:1796–1800). Here we resolve these apparently conflicting results and provide compelling in vivo evidence that Ste24p indeed functions at two steps of a-factor maturation using two methods. First, direct analysis of a-factor biosynthetic intermediates in the double mutant (ste24Δ rce1Δ) reveals a previously undetected species (P0*) that fails to be COOH terminally processed, consistent with redundant roles for Ste24p and Rce1p in COOH-terminal CAAX processing. Whereas a-factor maturation appears relatively normal in the rce1Δ single mutant, the ste24Δ single mutant accumulates an intermediate that is correctly COOH terminally processed but is defective in cleavage of the NH₂-terminal extension, demonstrating that Ste24p is also involved in NH2-terminal processing. Together, these data indicate dual roles for Ste24p and a single role for Rce1p in a-factor processing. Second, by using a novel set of ubiquitin–a-factor fusions to separate the NH₂- and COOH-terminal processing events of a-factor maturation, we provide independent evidence for the dual roles of Ste24p. We also report here the isolation of the human (Hs) Ste24p homologue, representing the first human CAAX protease to be cloned. We show that Hs Ste24p complements the mating defect of the yeast double mutant (ste24Δ rce1Δ) strain, implying that like yeast Ste24p, Hs Ste24p can mediate multiple types of proteolytic events.     

The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor

Saccharomyces cerevisiae Ste24p is a multispanning membrane protein implicated in the CAAX proteolysis step that occurs during biogenesis of the prenylated a-factor mating pheromone. Whether Ste24p acts directly as a CAAX protease or indirectly to activate a downstream protease has not yet been established. In this study, we demonstrate that purified, detergent-solubilized Ste24p directly mediates CAAX proteolysis in a zinc-dependent manner. We also show that Ste24p mediates a separate proteolytic step, the first NH(2)-terminal cleavage in a-factor maturation. These results establish that Ste24p functions both as a bona fide COOH-terminal CAAX protease and as an a-factor NH(2)-terminal protease. Importantly, this study is the first to directly demonstrate that a eukaryotic multispanning membrane protein can possess intrinsic proteolytic activity.     

A Novel Role of the Yeast CaaX Protease Ste24 in Chitin Synthesis

Ste24 is a membrane-integral CaaX metalloprotease residing in the endoplasmic reticulum (ER). In yeast, the only known substrate of Ste24 is the mating factor a precursor. A global screening for protein–protein interactions indicated that Ste24 interacts with chitin synthesis deficient (Chs)3, an enzyme required for chitin synthesis. We confirmed this interaction by yeast two-hybrid analyses and mapped the interacting cytoplasmic domains. Next, we investigated the influence of Ste24 on chitin synthesis. In sterile (ste)24Δ mutants, we observed resistance to calcofluor white (CFW), which was also apparent when the cells expressed a catalytically inactive version of Ste24. In addition, ste24Δ cells showed a decrease in chitin levels and Chs3-green fluorescent protein localized less frequently at the bud neck. Overexpression of STE24 resulted in hypersensitivity to CFW and a slight increase in chitin levels. The CFW phenotype of ste24Δ cells could be rescued by its human and insect orthologues. Although Chs3 binds to Ste24, it seems not to be a substrate for this protease. Instead, our data suggest that Chs3 and Ste24 form a complex in the ER that facilitates protease action on prenylated Chs4, a known activator of Chs3 with a C-terminal CaaX motif, leading to a more efficient localization of Chs3 at the plasma membrane.     

Kar9p Is a Novel Cortical Protein Required for Cytoplasmic Microtubule Orientation in Yeast

kar9 was originally identified as a bilateral karyogamy mutant, in which the two zygotic nuclei remained widely separated and the cytoplasmic microtubules were misoriented (Kurihara, L.J., C.T. Beh, M. Latterich, R. Schekman, and M.D. Rose. 1994. J. Cell Biol. 126:911–923.). We now report a general defect in nuclear migration and microtubule orientation in kar9 mutants. KAR9 encodes a novel 74-kD protein that is not essential for life. The kar9 mitotic defect was similar to mutations in dhc1/dyn1 (dynein heavy chain gene), jnm1, and act5. kar9Δ dhc1Δ, kar9Δ jnm1Δ, and kar9Δ act5Δ double mutants were synthetically lethal, suggesting that these genes function in partially redundant pathways to carry out nuclear migration. A functional GFP-Kar9p fusion protein localized to a single dot at the tip of the shmoo projection. In mitotic cells, GFP-Kar9p localized to a cortical dot with both mother–daughter asymmetry and cell cycle dependence. In small-budded cells through anaphase, GFP-Kar9p was found at the tip of the growing bud. In telophase and G1 unbudded cells, no localization was observed. By indirect immunofluorescence, cytoplasmic microtubules intersected the GFP-Kar9p dot. Nocodazole experiments demonstrated that Kar9p's cortical localization was microtubule independent. We propose that Kar9p is a component of a cortical adaptor complex that orients cytoplasmic microtubules.     

Liz1p, a Novel Fission Yeast Membrane Protein, Is Required for Normal Cell Division When Ribonucleotide Reductase Is Inhibited

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1⁺, which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1⁻ cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1⁺ encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.     

Role for the Ubiquitin-Proteasome System in the Vacuolar Degradation of Ste6p, the a-Factor Transporter in Saccharomyces cerevisiae

Ste6p, the a-factor transporter in Saccharomyces cerevisiae, is a multispanning membrane protein with 12 transmembrane spans and two cytosolic ATP binding domains. Ste6p belongs to the ATP binding cassette (ABC) superfamily and provides an excellent model for examining the intracellular trafficking of a complex polytopic membrane protein in yeast. Previous studies have shown that Ste6p undergoes constitutive endocytosis from the plasma membrane, followed by delivery to the vacuole, where it is degraded in a Pep4p-dependent manner, even though only a small portion of Ste6p is exposed to the vacuolar lumen where the Pep4p-dependent proteases reside. Ste6p is known to be ubiquitinated, a modification that may facilitate its endocytosis. In the present study, we further investigated the intracellular trafficking of Ste6p, focusing on the role of the ubiquitin-proteasome machinery in the metabolic degradation of Ste6p. We demonstrate by pulse-chase analysis that the degradation of Ste6p is impaired in mutants that exhibit defects in the activity of the proteasome (doa4 and pre1,2). Likewise, by immunofluorescence, we observe that Ste6p accumulates in the vacuole in the doa4 mutant, as it does in the vacuolar protease-deficient pep4 mutant. One model consistent with our results is that the degradation of Ste6p, the bulk of which is exposed to the cytosol, requires the activity of both the cytosolic proteasomal degradative machinery and the vacuolar lumenal proteases, acting in a synergistic fashion. Alternatively, we discuss a second model whereby the ubiquitin-proteasome system may indirectly influence the Pep4p-dependent vacuolar degradation of Ste6p. This study establishes that Ste6p is distinctive in that two independent degradative systems (the vacuolar Pep4p-dependent proteases and the cytosolic proteasome) are both involved, either directly or indirectly, in the metabolic degradation of a single substrate.     

The Class V Myosin Myo2p Is Required for Fus2p Transport and Actin Polarization during the Yeast Mating Response

Mating yeast cells remove their cell walls and fuse their plasma membranes in a spatially restricted cell contact region. Cell wall removal is dependent on Fus2p, an amphiphysin-associated Rho-GEF homolog. As mating cells polarize, Fus2p-GFP localizes to the tip of the mating projection, where cell fusion will occur, and to cytoplasmic puncta, which show rapid movement toward the tip. Movement requires polymerized actin, whereas tip localization is dependent on both actin and a membrane protein, Fus1p. Here, we show that Fus2p-GFP movement is specifically dependent on Myo2p, a type V myosin, and not on Myo4p, another type V myosin, or Myo3p and Myo5p, type I myosins. Fus2p-GFP tip localization and actin polarization in shmoos are also dependent on Myo2p. A temperature-sensitive tropomyosin mutation and Myo2p alleles that specifically disrupt vesicle binding caused rapid loss of actin patch organization, indicating that transport is required to maintain actin polarity. Mutant shmoos lost actin polarity more rapidly than mitotic cells, suggesting that the maintenance of cell polarity in shmoos is more sensitive to perturbation. The different velocities, differential sensitivity to mutation and lack of colocalization suggest that Fus2p and Sec4p, another Myo2p cargo associated with exocytotic vesicles, reside predominantly on different cellular organelles.     

The Sec63p J-Domain Is Required for ERAD of Soluble Proteins in Yeast

How misfolded proteins are exported from the ER to the cytosol for degradation (ER-associated Degradation, ERAD) and which proteins are participating in this process is not understood. Several studies using a single, leaky mutant indicated that Sec63p might be involved in ERAD. More recently, Sec63p was also found strongly associated with proteasomes attached to the protein-conducting channel in the ER membrane which presumably form part of the export machinery. These observations prompted us to reinvestigate the role of Sec63p in ERAD by generating new mutants which were selected in a screen monitoring the intracellular accumulation of the ERAD substrate CPY*. We show that a mutation in the DnaJ-domain of Sec63p causes a defect in ERAD, whereas mutations in the Brl, acidic, and transmembrane domains only affect protein import into the ER. Unexpectedly, mutations in the acidic domain which mediates interaction of Sec63p with Sec62p also caused defects in cotranslational import. In contrast to mammalian cells where SEC63 expression levels affect steady-state levels of multi-spanning transmembrane proteins, the sec63 J-domain mutant was only defective in ERAD of soluble substrates.     

Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor

CF II, a factor required for cleavage of the 3' ends of mRNA precursor in Saccharomyces cerevisiae, has been shown to contain four polypeptides. The three largest subunits, Cft1/Yhh1, Cft2/Ydh1, and Brr5/Ysh1, are homologs of the three largest subunits of mammalian cleavage-polyadenylation specificity factor (CPSF), an activity needed for both cleavage and poly(A) addition. In this report, we show by protein sequencing and immunoreactivity that the fourth subunit of CF II is Pta1, an essential 90-kDa protein originally implicated in tRNA splicing. Yth1, the yeast homolog of the CPSF 30-kDa subunit, is not detected in this complex. Extracts prepared from pta1 mutant strains are impaired in the cleavage and the poly(A) addition of both GAL7 and CYC1 substrates and exhibit little processing activity even after prolonged incubation. However, activity is efficiently rescued by the addition of purified CF II to the defective extracts. Extract from a strain with a mutation in the CF IA subunit Rna14 also restored processing, but extract from a brr5-1 strain did not. The amounts of Pta1 and other CF II subunits are reduced in pta1 strains, suggesting that levels of the subunits may be coordinately regulated. Coimmunoprecipitation experiments indicate that the CF II in extract can be found in a stable complex containing Pap1, CF II, and the Fip1 and Yth1 subunits of polyadenylation factor I. While purified CF II does not appear to retain the association with these other factors, this larger complex may be the form recruited onto pre-mRNA in vivo. The involvement of Pta1 in both steps of mRNA 3'-end formation supports the conclusion that CF II is the functional homolog of CPSF.     

Peb1p (Pas7p) is an intraperoxisomal receptor for the NH2-terminal, type 2, peroxisomal targeting sequence of thiolase: Peb1p itself is targeted to peroxisomes by an NH2-terminal peptide

Peb1 is a peroxisome biogenesis mutant isolated in Saccharomyces cerevisiae that is selectively defective in the import of thiolase into peroxisomes but has a normal ability to package catalase, luciferase and acyl-CoA oxidase (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359). Thiolase differs from these other peroxisomal proteins in that it is targeted by an NH2-terminal, 16- amino acid peroxisomal targeting sequence type 2 (PTS 2). This phenotype suggests that the PEB1 protein might function as a receptor for the PTS2. The PEB1 gene has been cloned by functional complementation. It encodes a 42,320-D, hydrophilic protein with no predicted transmembrane segment. It contains six WD repeats that comprise the entire protein except for the first 55 amino acids. Peb1p was tagged with hemagglutinin epitopes and determined to be exclusively within peroxisomes by digitonin permeabilization, immunofluorescence, protease protection and immuno-electron microscopy (Zhang, J. W., and P. B. Lazarow. 1995. J. Cell Biol. 129:65-80). Peb1p is identical to Pas7p (Marzioch, M., R. Erdmann, M. Veenhuis, and W.-H. Kunau. 1994. EMBO J. 13: 4908-4917). We have now tested whether Peb1p interacts with the PTS2 of thiolase. With the two-hybrid assay, we observed a strong interaction between Peb1p and thiolase that was abolished by deleting the first 16 amino acids of thiolase. An oligopeptide consisting of the first 16 amino acids of thiolase was sufficient for the affinity binding of Peb1p. Binding was reduced by the replacement of leucine with arginine at residue five, a change that is known to reduce thiolase targeting in vivo. Finally, a thiolase-Peb1p complex was isolated by immunoprecipitation. To investigate the topogenesis of Peb1p, its first 56-amino acid residues were fused in front of truncated thiolase lacking the NH2-terminal 16-amino acid PTS2. The fusion protein was expressed in a thiolase knockout strain. Equilibrium density centrifugation and immunofluorescence indicated that the fusion protein was located in peroxisomes. Deletion of residues 6-55 from native Peb1p resulted in a cytosolic location and the loss of function. Thus the NH2-terminal 56-amino acid residues of Peb1p are necessary and sufficient for peroxisomal targeting. Peb1p is found in peroxisomes whether thiolase is expressed or not. These results suggest that Peb1p (Pas7p) is an intraperoxisomal receptor for the type 2 peroxisomal targeting signal.     

A Novel Fission Yeast Gene, tht1+, Is Required for the Fusion of Nuclear Envelopes during Karyogamy

We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1⁺ gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1⁺ gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei.     

The Yeast Spindle Pole Body Component Spc72p Interacts with Stu2p and Is Required for Proper Microtubule Assembly

We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB). Here we report the identification of Spc72p, a protein that interacts with Stu2p. Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts. Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex. Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p. Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo. A temperature-sensitive spc72 mutation causes defects in SPB morphology. In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB. spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur. The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs.     

Erp1p and Erp2p, Partners for Emp24p and Erv25p in a Yeast p24 Complex

Six new members of the yeast p24 family have been identified and characterized. These six genes, named ERP1-ERP6 (for Emp24p- and Erv25p-related proteins) are not essential, but deletion of ERP1 or ERP2 causes defects in the transport of Gas1p, in the retention of BiP, and deletion of ERP1 results in the suppression of a temperature-sensitive mutation in SEC13 encoding a COPII vesicle coat protein. These phenotypes are similar to those caused by deletion of EMP24 or ERV25, two previously identified genes that encode related p24 proteins. Genetic and biochemical studies demonstrate that Erp1p and Erp2p function in a heteromeric complex with Emp24p and Erv25p.     

Mdm12p, a Component Required for Mitochondrial Inheritance That Is Conserved between Budding and Fission Yeast

Saccharomyces cerevisiae cells lacking the MDM12 gene product display temperature-sensitive growth and possess abnormally large, round mitochondria that are defective for inheritance by daughter buds. Analysis of the wild-type MDM12 gene revealed its product to be a 31-kD polypeptide that is homologous to a protein of the fission yeast Schizosaccharomyces pombe. When expressed in S. cerevisiae, the S. pombe Mdm12p homolog conferred a dominant-negative phenotype of giant mitochondria and aberrant mitochondrial distribution, suggesting partial functional conservation of Mdm12p activity between budding and fission yeast. The S. cerevisiae Mdm12p was localized by indirect immunofluorescence microscopy and by subcellular fractionation and immunodetection to the mitochondrial outer membrane and displayed biochemical properties of an integral membrane protein. Mdm12p is the third mitochondrial outer membrane protein required for normal mitochondrial morphology and distribution to be identified in S. cerevisiae and the first such mitochondrial component that is conserved between two different species.     

Integrin-mediated Activation of Focal Adhesion Kinase Is Required for Signaling to Jun NH2-terminal Kinase and Progression through the G1 Phase of the Cell Cycle

The extracellular matrix exerts a stringent control on the proliferation of normal cells, suggesting the existence of a mitogenic signaling pathway activated by integrins, but not significantly by growth factor receptors. Herein, we provide evidence that integrins cause a significant and protracted activation of Jun NH2-terminal kinase (JNK), while several growth factors cause more modest or no activation of this enzyme. Integrin-mediated stimulation of JNK required the association of focal adhesion kinase (FAK) with a Src kinase and p130(CAS), the phosphorylation of p130(CAS), and subsequently, the recruitment of Crk. Ras and PI-3K were not required. FAK-JNK signaling was necessary for proper progression through the G1 phase of the cell cycle. These findings establish a role for FAK in both the activation of JNK and the control of the cell cycle, and identify a physiological stimulus for JNK signaling that is consistent with the role of Jun in both proliferation and transformation.     

Identification of Proteassemblin,a Mammalian Homologue of the Yeast Protein,Ump1p,That Is Required for Normal Proteasome Assembly

We have identified a mammalian homologue of yeast Ump1p by searching for similar proteins in human and mouse expressed sequence tag (EST) databases. Ump1p is an accessory protein that is required for normal proteasome assembly in yeast (1). A mammalian homologue, which we refer to as “proteassemblin,” is a constituent of proteasome assembly intermediates (preproteasomes), but not fully assembled 20S proteasomes, as is Ump1p in yeast. We also provide evidence that proteassemblin is a constituent of pre-immunoproteasomes that contain the precursor of the interferon-γ-inducible subunit LMP2. By analogy with Ump1p, we hypothesize that proteassemblin is required for normal mammalian proteasome assembly.     

Sec35p,a Novel Peripheral Membrane Protein,Is Required for ER to Golgi Vesicle Docking

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE–associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.Protein transport through the secretory pathway occurs via transport vesicles under the direction of a large set of protein components (Rothman, 1994). The process can be divided into three stages: (a) vesicle budding, (b) vesicle docking, and (c) membrane fusion, with distinct sets of proteins mediating each phase. The budding step involves recruitment of coat proteins to the membrane and culminates with the release of coated vesicles (Schekman and Orci, 1996). The docking reaction is likely to require a set of integral membrane proteins on the vesicle and target membranes, termed v-SNAREs¹ and t-SNAREs (vesicle- and target membrane-soluble N-ethylmaleimide–sensitive fusion protein [NSF] attachment protein [SNAP] receptors, respectively), that are thought to confer specificity through their pair-wise interactions (Söllner et al., 1993b ). Small GTP-binding proteins of the rab family also assist in the docking process (Ferro-Novick and Novick, 1993), but their precise function is not known. The fusion step ensues after docking and results in the delivery of the vesicular cargo to the next compartment in the secretory pathway.Vesicular transport from the ER to the Golgi apparatus in the yeast Saccharomyces cerevisiae has been extensively characterized. Transport vesicle budding involves the assembly of the COPII coat, composed of the Sec13p/Sec31p (Pryer et al., 1993; Salama et al., 1993; Barlowe et al., 1994) and Sec23p/Sec24p heterodimers (Hicke and Schekman, 1989; Hicke et al., 1992), under the direction of an integral membrane protein, Sec12p (Nakano et al., 1988; Barlowe and Schekman, 1993), a small GTPase, Sar1p (Nakano and Muramatsu, 1989), and a multidomain protein, Sec16p (Espenshade et al., 1995; Shaywitz et al., 1997). Docking is thought to require a tethering event mediated by Uso1p (Cao et al., 1998), the yeast homologue of mammalian p115 (Barroso et al., 1995; Sapperstein et al., 1995), followed by or concurrent with the interaction of a set of ER to Golgi v-SNAREs, Bet1p, Bos1p, Sec22p (Newman and Ferro-Novick, 1987; Newman et al., 1990; Ossig et al., 1991; Shim et al., 1991; Søgaard et al., 1994) and perhaps Ykt6p (Søgaard et al., 1994; McNew et al., 1997), with the cognate t-SNARE on the Golgi, Sed5p (Hardwick and Pelham, 1992). For some time it was thought that fusion may be initiated by disassembly of the v/t-SNARE complex (Söllner et al., 1993a ) by yeast SNAP, Sec17p, (Griff et al., 1992) and NSF, Sec18p (Eakle et al., 1988; Wilson et al., 1989). However, this concept has been challenged by studies with a yeast system that reconstitutes homotypic vacuolar fusion, which suggests the action of Sec18p is before vesicle docking (Mayer et al., 1996; Mayer and Wickner, 1997). In addition, a prefusion role for NSF has been supported by the recent finding that liposomes bearing SNAREs alone can fuse in the absence of NSF (Weber et al., 1998).Several proteins involved in the regulation of yeast ER to Golgi v/t-SNARE complex assembly have been identified, including Ypt1p, Uso1p, and Sly1p. Ypt1p is a member of the rab family of small GTP-binding proteins that have been identified as important components of almost every stage in the secretory pathway (Ferro-Novick and Novick, 1993). Hydrolysis of GTP by rab-like proteins has been hypothesized to provide the regulatory switch that controls the fidelity of vesicular transport (Bourne, 1988). A second protein, Uso1p (Nakajima et al., 1991), appears to function in the same pathway as Ypt1p (Sapperstein et al., 1996), and both proteins have been demonstrated to be essential for SNARE complex assembly (Søgaard et al., 1994; Sapperstein et al., 1996; Lupashin and Waters, 1997). The third protein, Sly1p, is associated with the t-SNARE Sed5p (Søgaard et al., 1994). SLY1 is an essential gene in yeast (Dascher et al., 1991; Ossig et al., 1991), and Sly1p is required for ER to Golgi transport in vitro (Lupashin et al., 1996) and in vivo (Ossig et al., 1991). However, several lines of evidence, particularly from Sly1p homologues in other organisms, indicate that Sly1p may also function as a negative regulator of v/t-SNARE complex assembly, perhaps by preventing the association of the v- and t-SNAREs (Hosono et al., 1992; Pevsner et al., 1994; Schulze et al., 1994). A dominant allele of SLY1, termed SLY1-20, is capable of suppressing mutations in YPT1 and USO1, including complete deletions (Dascher et al., 1991; Sapperstein et al., 1996). Thus, in the presence of Sly1-20p, two components required for SNARE complex assembly are no longer essential. We have proposed a model (Sapperstein et al., 1996; Lupashin and Waters, 1997) in which Ypt1p and Uso1p function to relieve the inhibitory action of Sly1p on SNARE complex assembly. In this model Sly1-20p can be thought of as a noninhibitory form of SLY1 that renders Ypt1p and Uso1p superfluous.We believe that the ability of SLY1-20 to suppress defects in upstream docking regulators can be used to identify additional components involved in the regulation of vesicular docking. We have undertaken a genetic screen (to be presented elsewhere) to isolate novel components in this pathway which, when mutated, depend upon Sly1-20p for viability. In the course of this work, we discovered that two recently identified mutants, sec34 and sec35, can be suppressed by SLY1-20 and thus satisfy the criterion of our screen. These mutants were isolated in a novel screen to identify components involved in transport at any step between the ER and the trans-Golgi network (i.e., the Kex2p compartment) in yeast (Wuestehube et al., 1996). Both sec34 and sec35 accumulate the core-glycosylated form of secretory proteins at the nonpermissive temperature, indicating a block in ER to Golgi transport. Furthermore, electron microscopy indicated that both sec34 and sec35 accumulate numerous vesicles upon shift to the restrictive temperature (Wuestehube et al., 1996), a hallmark of genes whose protein products are involved in the docking or fusion phase of transport (Kaiser and Schekman, 1990). In this report we describe the cloning of SEC35 and analysis of its genetic interactions with other secretory genes. Strong genetic interaction between SEC35 and SLY1, YPT1, and USO1 suggests that Sec35p may function in vesicle docking. To test this possibility, we devised an in vitro transport assay that depends on the addition of purified Sec35p and Uso1p. Vesicles synthesized in the absence of functional Sec35p do not fuse with the Golgi compartment and remain as freely diffusible intermediates. Upon addition of Sec35p and Uso1p, vesicles dock to the Golgi and proceed to membrane fusion. Requirements for Sec35p at the vesicle docking step correlates our genetic experiments with the biochemically distinguishable steps of vesicle docking and membrane fusion.     

An NH2-Terminal Multi-Basic RKR Motif Is Required for the ATP-Dependent Regulation of hIK1

We previously demonstrated that the ATP/PKA?dependent activation of the human intermediate conductance, Ca²⁺?activated K⁺ channel, hIK1, is dependent upon a C?terminal motif. The NH₂?terminus of hIK1 contains a multi?basic ¹³RRRKR¹⁷ motif, known to be important in the trafficking and function of ion channels. While individual mutations within this domain have no effect on channel function, the triple mutation (¹⁵RKR¹⁷/AAA), as well as additional double mutations, result in a near complete loss of functional channels, as assessed by whole?cell patch?clamp. However, cell?surface -immunoprecipitation studies confirmed expression of these mutated channels at the plasma membrane. To elucidate the functional consequences of the ¹⁵RKR¹⁷/AAA mutation we performed inside?out patch clamp recordings where we observed no difference in Ca²⁺ affinity between the wild?type and mutated channels. However, in contrast to wild?type hIK1, channels expressing the ¹⁵RKR¹⁷/AAA mutation exhibited rundown, which could not be reversed by the addition of ATP. Wild-type hIK1 channel activity was reduced by alkaline phosphatase both in the presence and absence of ATP, indicative of a phosphorylation event, whereas the ¹⁵RKR¹⁷/AAA mutation eliminated this effect of alkaline phosphatase. Further, single channel analysis demonstrated that the ¹⁵RKR¹⁷/AAA mutation resulted in a four?fold lower channel open probability (P_o), in the presence of saturating Ca²⁺ and ATP, compared to wild?type hIK1. In conclusion, these results represent the first demonstration for a role of the NH₂?terminus in the second messenger?dependent regulation of hIK1 and, in -combination with our previous findings, suggest that this regulation is dependent upon a close NH₂/C?terminal association.