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Vol. 14, Issue 12, 4971-4983, December 2003
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Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235-1634
Submitted July 2, 2003;
Revised July 31, 2003;
Accepted August 1, 2003
Monitoring Editor: Benjamin Glick
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The APT subfamily contains five genes, DRS2, DNF1, DNF2, DNF3, and NEO1, that share 40-83% amino acid sequence similarity with each other (Catty et al., 1997; Hua et al., 2002). APTs, or flippases, couple ATP hydrolysis to the translocation of phosphatidylserine (PS) and/or phosphatidylethanolamine (PE) from the external leaflet of a membrane bilayer to the cytosolic leaflet and appear to be responsible for concentrating PS and PE in the cytosolic leaflet of biological membranes (Balasubramanian and Schroit, 2003). This activity has been primarily defined in the plasma membrane of blood cells (Seigneuret and Devaux, 1984; Auland et al., 1994) and in bovine chromaffin granule membranes (Zachowski et al., 1989). The latter APT appears to be equivalent to ATPase II, which is 47% identical to Drs2p from yeast (Tang et al., 1996). The human genome encodes multiple members of the APT subfamily including genes implicated in genetic disorders. FIC1 mutations cause a defect in bile secretion and ATP10C is in an imprinted region of chromosome 18 linked to Angelman syndrome and potentially autism (Thompson and Jansen, 2000 Herzing et al., 2001; Meguro et al., 2001). In addition, the mouse ATP10C gene is linked to an increased body fat phenotype (Dhar et al., 2000).
The localization and cellular requirements for the yeast APTs are starting to be defined. Drs2p and Dnf3p localize to late Golgi and perhaps endosomal membranes, whereas Dnf1p and Dnf2p localize primarily to the plasma membrane (Chen et al., 1999; Hua et al., 2002; Pomorski et al., 2003). Deletion of all four of these genes is lethal in yeast, indicating that DRS2 and the DNFs form an essential gene group (Hua et al., 2002). Strains harboring disruptions of DNF1,2 or DRS2 exhibit a defect in lipid translocation across the plasma membrane and expose more PE on the external leaflet than wild-type cells (Tang et al., 1996; Gomes et al., 2000; Pomorski et al., 2003). These mutant phenotypes are consistent with the proposed translocase activity for the Drs2/Neo1 family ATPases, although this conclusion has been controversial (Siegmund et al., 1998; Marx et al., 1999) and direct evidence that these proteins catalyze lipid translocation is still lacking.
Drs2 and Dnf proteins are also required for protein trafficking between the Golgi complex, plasma membrane, and endosomal/vacuolar system (Chen et al., 1999; Gall et al., 2002; Hua et al., 2002; Pomorski et al., 2003). DRS2 interacts genetically with clathrin and ARF, and drs2
exhibits defects in late Golgi function that are similar to clathrin mutants (Chen et al., 1999). In addition, drs2 mutants also exhibit a defect in generating a specific class of exocytic vesicles carrying invertase and acid phosphatase (Gall et al., 2002). These vesicles require clathrin for their formation and at least a portion of them are clathrin-coated. The drs2 dnf1 double mutant exhibits a significantly stronger defect in the alkaline phosphatase and carboxypeptidase Y (CPY) vacuolar transport pathways compared with the single mutants. This suggests that Drs2p and Dnf1p are functionally redundant in their ability to support vacuolar protein transport, whereas only Drs2p can support the generation of exocytic vesicles. Moreover, the dnf1 dnf2 dnf3 triple deletion mutant exhibits a defect in an endosome to TGN recycling pathway traveled by a v-SNARE protein (Hua et al., 2002). ARF, clathrin, and/or adaptins (AP-1, AP-3 and GGAs) have been implicated in each of these pathways that require Drs2/Dnf proteins for normal function.
Among the five potential APTs in yeast, NEO1 is unique in that deletion of this gene alone is lethal (Prezant et al., 1996), and so none of the other APT subfamily members can perform the essential function of Neo1p. In addition, overexpression of NEO1 cannot suppress the drs2 cold-sensitive growth defect, nor can overexpression of DRS2 rescue neo1 lethality (Hua et al., 2002). NEO1 was first identified in a screen for genes that confer resistance to the aminoglycoside neomycin upon overexpression (Prezant et al., 1996). Neo1p overexpression did not confer resistance to other drugs tested including ethidium bromide, cycloheximide, and chloramphenicol, indicating that Neo1p is not a multidrug resistance protein. The Neo1p ATPase activity is required to support cell viability and to confer neomycin resistance (Prezant et al., 1996). It is not known if Neo1p can directly pump neomycin out of the cell or if it confers the drug resistance through another mechanism. Nothing is known about the essential cellular functions of Neo1p.
Because Drs2p and Dnf proteins are involved in protein transport in the late secretory and endosomal pathways, we hypothesized that Neo1p might also be required for protein transport in the secretory pathway. To test this possibility, conditional alleles of neo1 were generated and their effect on protein transport was determined. We found that Neo1p is indeed required for efficient protein transport in the early secretory pathway and Golgi-dependent glycosylation. Protein transport between the ER and Golgi is mediated by small transport vesicles, with COPII-coated vesicles carrying cargo in the anterograde direction and COPI-coated vesicles carrying cargo in the retrograde direction (Kirchhausen, 2000). Temperature-sensitive mutations in COPII subunits (e.g., sec12 or sec23) cause a block in the ER-to-Golgi transport and the accumulation of all cargo proteins in the ER at the nonpermissive temperature (Barlowe, 2002). COPI mutations (e.g., sec21) perturb retrograde transport and can also cause a defect in ER-to-Golgi transport, although not all proteins are subject to this block. For example, Hsp150 and invertase are secreted efficiently from sec21 cells, whereas carboxypeptidase Y (CPY) and pro–
-factor are blocked in the ER (Gaynor and Emr, 1997). Mutations in early Golgi ARF GAPs (Glo3 and Gcs1) also perturbs CPY transport and invertase glycosylation but not invertase secretion (Poon et al., 1999). Rer1p can be used to distinguish a primary defect in anterograde or retrograde transport, because this protein is trapped in the ER in COPII mutants but mislocalized to the vacuole in COPI mutants (Sato et al., 2001). The neo1 mutants also exhibit cargo-selective defects in anterograde protein transport and mislocalize Rer1p to the vacuole. These phenotypes suggest that Neo1p is required for COPI-dependent retrograde transport from the Golgi to the ER.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Yeast strains used in this study are listed in Table 1. The yeast knockout strain collection was originally purchased from Research Genetics, which is now Resgen, Invitrogen Corporation (Carlsbad, CA). ZHY9075E was generated by sporulation of BY4743 neo1 pBM743-NEO1 (Hua et al., 2002). ZHY219RR and all ZHY628 strains were generated by replacing pBM743-NEO1 in ZHY9075E by either p413-NEO1 (carrying wild-type NEO1) or p413-neo1-1 through 6 (carrying neo1 ts alleles).
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The plasmid p413-NEO1PM was generated by replacing the ADH promoter from p413-ADH with the PCR-amplified NEO1 promoter region (518 bp) on a SacI/XbaI fragment. The NEO1 coding sequence from pYGW1-NEO1 (Hua et al., 2002) was cloned into the EcoRI/SalI site of p413-NEO1PM to generate p413-NEO1. Sequencing of p413-NEO1 revealed two nucleotide changes compared with published sequence and causes two amino acid changes (V186M and T558A). This plasmid fully rescues the neo1 lethality phenotype. Other plasmids used in this study are pSKY5/RER1-0 (a cen-based URA3 plasmid carrying Rer1-GFP; Sato et al., 2001), pOH-URA3 (a 2-µm plasmid with OCH1-HA; Harris and Waters, 1996), and pRS426-MNN1 (Graham et al., 1994).
Isolation of neo1-ts Alleles
Random PCR mutagenesis (with 3.5 mM MgCl2, 0.5 mM MnCl2, 0.45 mM dATP, 0.72 mM dCTP, 0.16 mM dGTP, and 1.12 mM dTTP in the PCR reaction for 35 cycles) was used to generate mutations in the NEO1 gene. Initial attempts at mutagenesis using higher concentrations of dNTPs and fewer cycle numbers failed to yield a neo1-ts allele. Primers used for PCR were 5'-TTTGTGCCAACCCTATTATATGAA, and 5'-GCATTGTTTTATTCCATGTGTAGA. Plasmid p413-NEO1 was gapped using NcoI and StuI and was cotransformed with the mutagenized PCR products into ZHY9075E. The resulting transformants (
10,000) were then replicated onto 5-fluoroorotic acid (5-FOA) plates to select against the original wild-type NEO1 plasmid (pBM743-NEO1 harboring URA3). Approximately 1000 colonies grew on the 5-FOA plates, which were then tested for growth at 37°C. Six colonies exhibited a tight ts growth defect at 37°C. Plasmids were rescued from these six strains and named p413-neo1-1 to p413-neo1-6. The following mutations are found in the six neo1 ts alleles: neo1-1, D356V, I381V, A420V, E528D, T529P, Q538L, D568V, T604N, Q727R, R805K, E807K, T1043S; neo1-2, H346L, A431V, V457I, L465I, P491L, S502C, S542C, P581L, S618T, K672E, L719W, D725V, I732N, L752S, Q812R, A854V; neo1-3, H279Q, T337I, A370V, A375S, V402A, V430L, N437I, R496H, M569K; neo1-4, M234I, S297T, C319S, N366I, E528K, R565H, E624D, V674A, D716E, S729N, M872K; neo1-5, A370V, P491Q, S542R, G698S, S908T, A940V; neo1-6, C380Y, F661S, C855S, A951T, Y957N, V986I, H993L, T1000S.
Integration of neo1-ts Alleles into the Yeast Genome
To facilitate genetic crosses of neo1-ts mutants with other mutants, neo1-ts alleles were integrated into the NEO1 locus. The integrating neo1-ts plasmids (pZH1125-15B and pZH1125-34A for neo1-1 and neo1-2, respectively) were made by first inserting the SacI/SalI fragment from p413-neo1-1 or p413-neo1-2 into pRS303, and then a 600-bp PCR-amplified KanMX fragment was inserted into the ApaI/XhoI site of the resulting plasmid. pZH1125-15B and pZH1125-34A were then cut with NruI, a unique restriction enzyme site located within the KanMX fragment, and then transformed into BY4743 neo1::KanMX, to generate the BY4743 neo1::neo1-1 (or neo1-2) HIS3 strain. These strains were then subjected to sporulation, and the progeny were tested for ts growth to identify the integrated neo1-ts mutants (ZHY129-15B28A and ZHY129-34A2A). To integrate neo1 ts alleles into the SEY6210 background, a PCR-amplified KanMX-NEO1 knockout module (http://www-sequence.s-tanford.edu/group/yeast_deletion_project/deletions3.html) was first transformed into a SEY6210.5 diploid (Robinson et al., 1988). Then the neo1 ts integration plasmids pZH1125-15B and pZH1125-34A were transformed into the resulting strain. The transformants were then subjected to sporulation and tested for a ts growth phenotype to identify neo1-ts integrated mutants (All ZHY124 strains are in SEY6210 background).
Genetic Analysis of neo1 ts Mutants
Genetic crosses between neo1-ts (ZHY129-15B28A and ZHY129-34A2C) and arf1, glo3, gcs1, gda1, mnn9, dnf1, dnf2, and dnf2 strains were done in the BY4741/BY4742 strain background. Genetic crosses between neo1-ts (ZHY124-15B1B, ZHY124-15B1C, ZHY124-34A2A, and ZHY124-34A2B) and sec21-1, ret1-1, sec18-1, sec23-1, sec1-1, sec12-1, sec14-1, and sec7-4 were done in the SEY6210 strain background. Diploids from the above crosses were subjected to sporulation and tetrad dissection, and the double mutants were collected if available.
Immunological and Imaging Methods
Cell labeling, immunoprecipitation (Gaynor and Emr, 1997), and immunoblotting (Chen et al., 1999) were performed as described previously. Anti--factor (Graham and Emr, 1991) and anti-carboxypeptidase Y (CPY) serum (Klionsky et al., 1988) were used for immunoprecipitation. The 9E10 mouse monoclonal c-Myc antibody (Oncogene Research Products, Darmstadt, Germany) (1:100), polyclonal affinity-purified rabbit anti-HA antibody (Zymed Laboratories, South San Francisco, CA; 1:100), and anti-Mnn1p antibody (Graham et al., 1994; 1:50) were used for immunofluorescence microscopy. Immunofluorescence and green fluorescent protein (GFP) fluorescence were observed using an Axioplan microscope (Carl Zeiss, Thornwood, NY), and fluorescent images were processed using MetaMorph 4.5 software (Universal Imaging, Downingtown, PA). Samples for electron microscopy were prepared as described previously (Rieder et al., 1996) and sections (50-60 nm) were viewed on a CM12 electron microscope (Philips, Eindhoven, Netherlands).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Thus, it is possible that the c-myc epitope tag induced an artificially high concentration of Neo1-myc in the ER of our strain.
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Neo1p Depletion Causes Defects in Protein Transport and Golgi Glycosylation
A conditional allele of NEO1 was produced by cloning this gene under transcriptional control of the GAL1 promoter (Hua et al., 2002) so that its expression could be controlled by carbon source (Johnston and Davis, 1984). The neo1 GAL::NEO1 strain grew well on inducing galactose media, but did not form visible colonies on glucose media where Neo1p expression was repressed. After initial growth in the presence of galactose, 18 h were required for cell growth to slow down in YP glucose medium and 24 h in synthetic minimal glucose medium. This represented 10 generations for both media, which is typical for proteins that do not turn over rapidly.
To determine if Neo1p is involved in protein trafficking, cells depleted for Neo1p were pulse-labeled and chased to examine the kinetics of pro–-factor processing and transport (Figure 2). The yeast -factor mating pheromone is synthesized in the ER as a high-molecular-weight precursor and is further modified in the Golgi complex to produce the heterogeneously glycosylated pro–-factor form that migrates as a high-molecular-mass smear by SDS-PAGE. This precursor is processed in the TGN through a series of proteolytic events, initiated by Kex2p, to produce the mature -factor peptide, which is secreted (Fuller et al., 1988).
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The neo1 GAL::NEO1 strain was grown in SM galactose and shifted to SM glucose for 24 h to deplete Neo1p. Cells were then labeled with [35S]methionine/cysteine for 15 min and chased for the time points indicated. In cells maintained in galactose, pro–-factor was processed to the mature form very quickly (Figure 2, galactose), indicating that protein transport from the ER through the Golgi complex was efficient. However, for cells grown in glucose media, both the ER and Golgi forms of pro–-factor were present throughout the chase. In addition, the Golgi pro–-factor forms appeared to be substantially underglycosylated because most of this precursor migrated just above the ER form (Figure 2, glucose). This result indicates that depletion of Neo1p causes defects in protein transport from the ER to the Golgi and potentially through the Golgi complex. Depletion of Neo1p also appeared to perturb Golgi-dependent glycosylation.
Isolation of neo1-ts Mutants
To facilitate studies of its essential function, we screened for temperature sensitive (ts) alleles of NEO1. The middle three fourths of the NEO1 gene, including the ATPase domain and six flanking transmembrane domains, was targeted for mutagenesis (Figure 3 and MATERIALS AND METHODS). Among nearly 10,000 transformants screened, we found six colonies that grew well at 27°C but exhibited a tight ts growth defect. Three mutants could not form visible colonies at 34°C, and three could grow at 34°C but not 37°C (Figure 4, YPD). All six mutant alleles have been sequenced and each mutant contains from 6 to 16 missense mutations, with 10 mutations on average, spanning the targeted 810 amino acids (Figure 3). It does not appear that the P-type ATPase conserved motifs were more mutated than the other regions. Instead, it seems that the mutations clustered at the predicted transmembrane motifs, especially in transmembrane domain 3 (TMD3) where five of the six alleles carried mutations. Other clustered mutations included four residues that were mutated in two different alleles, and five sites where adjacent amino acids were mutated in the same or different alleles. The neo1-5 allele only contains six mutations, and three of them overlap with mutations from other alleles, suggesting that mutations of A370, P491, and S542 likely contribute to the ts phenotype (Figure 3). Another interesting phenomenon is that the mutations tend to cluster even within one allele. For example, five of eight mutations from neo1-6 reside within 50 of the total of 810 amino acids targeted. Eight of nine mutations in neo1-3 reside within the first third of this region (Figure 3). This phenomenon might reflect a destabilization of local secondary structures in the ts mutant proteins.
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The neo1-ts mutants we isolated can still divide a few times after shifting to the nonpermissive temperature. The neo1-ts mutants formed microcolonies containing a few to several dozens of cells when grown at 37°C and this varied depending on the allele. In addition, the growth rate of neo1-ts mutants in liquid media continued unperturbed for 6 h when cells were shifted from 27 to 37°C. These data indicate that either all of the neo1-ts alleles are slowly inactivated after temperature shift, that they never lose complete function at 37°C, or that cells can divide a few times in the absence of Neo1p function.
The ts Growth Defect of neo1-ts Mutants Is Suppressed by High Osmotic Support
The glycosylation defect observed by depleting Neo1p suggested that cell wall biosynthesis might be perturbed in neo1 mutants. Mutants that grow poorly because of a defective cell wall can be remedied by sorbitol or other osmotic support and all six neo1-ts mutants can be rescued by osmotic support at the nonpermissive temperature. The growth of most mutants improved significantly on 1 M sorbitol, and they grew nearly as well as wild-type on 1.5 M sorbitol (Figure 4 and Table 2). In addition, 1 M NaCl also suppressed the neo1 ts growth defect (Table 2). The yeast cell wall consists of glucans, mannans, and chitin (Klis et al., 2002). Many cell wall biosynthesis mutants are hypersensitive to the chitin-binding compound calcofluor white (CW). The neo1-ts mutants were also hypersensitive to CW (Table 2), further suggesting a cell wall defect.
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Interestingly, nonfermentable carbon sources like glycerol or ethanol also supported neo1-ts growth at the nonpermissive temperature (Figure 4 and Table 2), and even galactose partially supported neo1-ts growth (Table 2). One possible explanation for these results is that cells grow more slowly on the nonglucose carbon source, and this might allow for more time to assemble a functional cell wall. However, it is also possible that in nonglucose carbon source media, other pathways are activated to suppress the neo1-ts mutant phenotype. It is noteworthy that neither the high osmotic support, nor the nonglucose carbon sources were able to suppress neo1 lethality (Table 2), indicating that Neo1p is not solely involved in cell wall synthesis. This also indicates that despite the heavy mutagenesis, all of the neo1-ts alleles retain some function at 37°C; otherwise these mutants would fail to grow on sorbitol.
Because NEO1 confers resistance to neomycin when overexpressed, we tested the neo1-ts mutants for neomycin hypersensitivity. As predicted, most neo1-ts mutants are hypersensitive to neomycin (Table 2). Interestingly, neo1-6 is not nearly as sensitive to neomycin as the other mutants. This suggests that the different neo1 ts alleles might affect different functions of Neo1p. Neo1p is homologous to Drs2p, and drs2 has been shown to be hypersensitive to Mn2+, Ca2+, and other cations. Therefore, the neo1-ts mutants were also tested on the plates containing different cations at permissive temperature. The neo1-ts mutants are not significantly hypersensitive to any cation tested (Table 2). However, a few neo1 ts alleles did exhibit an increased sensitivity to NaF, an inhibitor of P-type ATPases (Table 2). Hypersensitivity to calcofluor white, neomycin, and NaF at the permissive growth temperature indicates that all of the neo1-ts alleles have partially lost function at 27°C. Consistent with this, we find that the neo1-ts cells grown at permissive temperature are enlarged and a small percentage of the cells are also elongated (Figure 5, A and B). This distinctive phenotype is not significantly aggravated when cells are shifted to the nonpermissive temperature. Interestingly, growth in 1.5 M sorbitol appeared to have partially suppressed the increased width of the cell, but severely exacerbated the hyperelongated morphology (Figure 5, A and B).
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Protein Transport and Glycosylation Defects Exhibited by neo1-ts
Strains harboring a strong (neo1-1) or weak (neo1-2) ts allele of NEO1 were examined for defects in protein transport and modification at the permissive and nonpermissive temperatures. Cells growing at 27°C, or shifted to 37°C for 1 or 3 h, were subjected to pulse-chase analysis of CPY and -factor transport. CPY is synthesized in the ER as the p1 precursor form and is modified on N-linked oligosaccharides by Golgi mannosyltransferases to form the p2 precursor. p2 CPY is sorted from secreted proteins in the late Golgi and is transported through a prevacuolar endosome to the vacuole where it is processed to the mature form (mCPY; Stevens et al., 1982; Vida et al., 1993).
At 27°C, the kinetics of transport and modification of CPY and -factor in the neo1 mutants was similar to wild-type cells, but a defect became apparent after incubation at 37°C (Figure 6). In wild-type cells preincubated at 37°C for 1 h, about half of the labeled CPY was in the p2 form at the beginning of the chase period (0 min), and most of the protein was converted to the mature form 10 min later. However, most of CPY was still in the ER form at 0 min in the neo1 mutants and the p2 form of CPY was underglycosylated, causing a faster migration in the gel so that it could not be clearly separated from the p1 form (Figure 6). For the "tighter" neo1-1 mutant, only half of CPY was converted to the mature form in 10 min. The transport and glycosylation defects became more apparent for both mutants after 3 h at 37°C.
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After 1 h of incubation at the nonpermissive temperature, the transport and modification of pro–-factor (immunoprecipitated from the same cell extracts as CPY) was surprisingly normal (Figure 6). A defect became apparent after a 3-h shift to the nonpermissive temperature in the neo1 mutants, with more ER form accumulating and less mature form present at the beginning of the chase. The fully glycosylated form of pro–-factor, which can be seen in the 37°C 1-h panel (Figure 6, lanes 9 and 11), was replaced by underglycosylated precursor in the 3 h neo1-ts samples (Figure 6, lanes 15 and 17). These results are consistent with what was observed in the Neo1p depletion experiments and suggest that Neo1p is required for efficient protein transport from the ER to the Golgi and for synthesis of the outer chain on N-linked oligosaccharides in the Golgi complex.
neo1-ts Exhibits COPI Mutant Phenotypes
Proteins are transported from the ER to the Golgi complex in COPII-coated transport vesicles. Temperature-sensitive COPII mutants (e.g., sec12 or sec23) exhibit a block in the ER-to-Golgi transport of all cargo proteins tested (Hicke and Schekman, 1989; Barlowe and Schekman, 1993). The differential effect of neo1-ts on CPY and -factor transport noted above is more consistent with a defect in Golgi-to-ER retrograde transport mediated by COPI. COPI mutants are known to exhibit cargo-specific defects in anterograde transport that is presumably a secondary effect of perturbing the recycling of cargo receptors (Gaynor and Emr, 1997).
To test if neo1 mutants exhibit a cargo-specific defect in protein transport, general secretion was examined in the neo1 ts mutants (Figure 7). Cells maintained at 27°C or shifted to 37°C for 2 or 3 h were labeled with [35S]methionine/cysteine. Cells and medium were separated by centrifugation, and proteins secreted into the medium were precipitated with TCA and subjected to SDS-PAGE. At 27°C, the general secretion of proteins into the media was about the same between the wild-type strains (WT and NEO1) and neo1 mutants. When cells were shifted to 37°C for 2 h, Hsp150, the top band on the gel migrating at about 150 kDa, was still secreted from both neo1 mutants, whereas lower molecular weight proteins were missing (Figure 7, 2 h). This pattern of secreted proteins is similar to the COPI mutant phenotype, in which only a subset of secreted proteins were affected at the nonpermissive temperature (Gaynor and Emr, 1997). When cells were shift to 37°C for 3 h, Hsp150 secretion also decreased in neo1-1 (Figure 7, 3 h), although neo1-2 still exhibited the cargo-specific defect in secretion. We also noticed that Hsp150 from neo1 ts mutants labeled at the nonpermissive temperature migrated slightly faster in gels than that from the wild-type cells. This suggests that Golgi O-glycosylation is partially defective in neo1, because Hsp150 is extensively O-glycosylated with no N-linked oligosaccharides.
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Rer1p is a protein that continuously cycles between the early Golgi complex and the ER, and is a sensitive marker to distinguish whether a mutant exhibits a primary defect in anterograde or retrograde transport (Sato et al., 2001). Sato et al. (2001) found that Rer1-GFP is primarily localized to the Golgi in wild-type cells, but is trapped in the ER in COP II mutants and mislocalized to the vacuole in COPI mutants. To test if Neo1p is involved in anterograde or retrograde transport between the ER and Golgi complex, Rer1-GFP was expressed in neo1-ts cells. At the permissive temperature, Rer1-GFP was mainly localized in punctate structures in both wild-type and neo1-1 cells, although a slightly more diffuse localization was noted for the neo1-1 cells (Figure 8, 27°C). After cells were shifted to nonpermissive temperature for 2 h, a significant amount of Rer1-GFP localized to vacuoles in neo1-1 cells (Figure 8, 37°C). Wild-type cells also exhibited more Rer1-GFP in the vacuole at 37°C, but not to the extent observed in neo1-1 cells. No ER accumulation of Rer1-GFP was found in the neo1-1 mutant. This result suggests that neo1 does not directly perturb the COPII-dependent anterograde pathway but does perturb a COPI-dependent retrograde transport pathway. Consistent with these results, we also find that neo1-ts cells secrete more Kar2p (an ER resident protein bearing the HDEL retrieval signal) than wild-type yeast (our unpublished results).
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Mutations affecting the same pathway will often show genetic interactions and so double mutants were constructed by crossing neo1-1 with several different mutants with defects in protein transport or glycosylation (Figure 9A). The viable double mutants were then tested for growth at several different temperatures. No interaction was observed between neo1-1 and the COPII alleles (sec12-4, sec23-1), but the neo1 COPI (sec21-1, ret1-1) double mutants grew slower on the tetrad dissection plates and showed an increase in temperature sensitivity relative to the single mutants (Figure 9C and our unpublished results). The strongest genetic interaction was observed between neo1-1 and drs2. Most of the double mutants produced from this cross failed to grow (Figure 9B), and the few that did survive grew extremely slowly. Although the drs2 neo1 synthetic lethality may be caused by the combined defects in clathrin and COPI pathways, it more likely reflects a common biochemical function for Neo1p and Drs2p in the Golgi complex. These proteins are 30% identical in amino acid sequence and are both in the APT family of P-type ATPases. No significant genetic interaction was observed between neo1-1 and arf1, ARF-GAPs (gcs1, glo3), or an ARF-GEF (sec7-4), which was surprising considering that both drs2 arf1 and drs2 neo1-ts are synthetically lethal. In addition, no genetic interaction was observed with mutations that perturb later steps in protein transport (sec14-3, sec1-1) or glycosylation (gda1, mnn9), or deletions of other APT family members (dnf1, dnf2; Figure 9A). Thus, although the genetic interaction found between neo1-1 and COPI alleles was modest, it was specific and supported a role for Neo1p in COPI function.
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The anterograde protein transport defect appeared more severe for cells depleted for Neo1p compared with the experiments using neo1-ts mutants. We suspected that the different temperatures used to assay protein transport might have contributed to these differences. Therefore, we labeled Neo1-depleted cells at 37°C to examine CPY and -factor transport at the higher temperature. CPY transport was approximately twofold faster for both wild-type and Neo1p-depleted cells at 37°C (compared with 30°C) but the transport defect was still apparent in the depleted cells (Figure 10, CPY). In contrast to CPY, the protein transport defect was significantly alleviated at 37°C for pro–-factor (Figure 10, 37°C). More ER form of pro–-factor was present at the beginning of the chase (time 0) in the Neo1-depleted cells than in wild-type, but this quickly chased to the mature form. These results are very similar to those obtained with the neo1-ts strains at 37°C and suggest that there is a greater demand for Neo1p function in recycling cargo receptors at lower temperatures.
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The neo1-1 Mutant Accumulates Abnormal Membrane Structures
Because the neo1 mutants exhibited significant defects in the secretory pathway, we examined these cells by electron microscopy (EM) to see if any organelle membrane or intermediate transport vesicles accumulate in this mutant. The appearance of neo1-1 cells grown at 27 and 37°C were quite different (Figure 11). At the permissive temperature, the neo1-1 mutant accumulated small vesicles (40.9 ± 9.2 nm in diameter) and lipid droplets (Figure 11, A and B) compared with wild-type cells. Vacuoles were also fragmented, which was confirmed by staining living cells with FM4–64 or CDC-FDA (our unpublished results). The fragmented vacuole phenotype was not completely penetrant, and cells with normal vacuoles could easily be found (see Figure 8). When neo1 was shifted to the nonpermissive temperature for 3 h, the number of small vesicles was substantially reduced, and several kinds of abnormal membrane structures were found to accumulate. Some cell sections contained multiple vacuole-like structures, although they did not stain darkly as do vacuoles in wild-type cells (Figure 11D, arrows). Other sections showed an accumulation of abnormal membrane structures that are likely enlarged Golgi cisternae (Figure 11E, arrows), and continuously looped membrane sheets, which are likely an expanded ER (Figure 11F, arrow). The accumulation of different abnormal membrane structures in neo1 indicates a defect in membrane trafficking through the secretory pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-factor transport from the ER to the Golgi and potentially through the Golgi complex. 3) Golgi-dependent modification of N-linked oligosaccharides on CPY and pro–-factor is perturbed upon Neo1p depletion. 4) The neo1-ts mutations cause these same protein transport and glycosylation defects at the nonpermissive temperature, albeit less severely, and seem to preferentially perturb CPY exit from the ER. 5) The neo1-ts mutants exhibit a block in the secretion of a subset of proteins, whereas others, such as Hsp150, are secreted efficiently. 6) Rer1-GFP, a protein that continuously cycles between the ER and Golgi, is mislocalized to the vacuole in neo1-ts. 7) The neo1-ts mutants accumulate small vesicles at the permissive temperature and accumulate ER and Golgi membranes at the non-permissive temperature.
The neo1 mutants exhibit several phenotypes in common with COPI mutants. These include a cargo-specific defect in secretion, aberrant glycosylation of cargo in the Golgi, and mislocalization of Rer1-GFP to the vacuole. Rer1p is an integral membrane protein required for retrograde transport of other membrane proteins to the ER. It has a C-terminal dilysine-like motif that binds COPI and links other cargo (Sec12p or Sec71p) to COPI via transmembrane domain interactions (Sato et al., 1997, 2001). Thus, Rer1p appears to be a cargo receptor for proteins retrieved from the Golgi to the ER in COPI vesicles and must continuously shuttle between the ER and Golgi to perform this task. COPII mutants accumulate Rer1-GFP in the ER and COPI mutants mislocalize Rer1-GFP to the vacuole (Sato et al., 2001). The mislocalization of Rer1-GFP to the vacuole of neo1-ts suggests that these cells have a primary defect in retrograde rather than anterograde transport. In addition, the sec21 (
-COPI) mutant perturbs anterograde transport of a subset of secreted proteins and the spectrum of affected and unaffected proteins is similar in the neo1 mutants (Gaynor and Emr, 1997). This is likely caused by a failure to recycle cargo receptors needed for anterograde transport of some proteins.
Although all COPI mutants appear to exhibit a defect in retrograde transport, not all display a defect in anterograde protein transport (Cosson et al., 1996; Duden et al., 1998). Thus, the modest anterograde transport defect observed in neo1-ts at 37°C does not indicate that the retrograde defect is marginal. In fact, the Neo1p requirement for anterograde transport appears less critical at higher temperatures because depletion of Neo1p at 30°C produced a stronger pro–-factor block than depletion at 37°C. Moreover, it is clear that none of the neo1-ts alleles cause a complete loss of function at the nonpermissive temperature. The ts growth defect of all of the neo1-ts mutants can be suppressed by osmotic support, but this treatment does not allow neo1 cells to grow at any temperature tested. Therefore, the protein trafficking defects observed in neo1-ts cells was caused by a partial loss of Neo1p function. Neo1p depletion caused a stronger defect in anterograde protein transport and even these experiments may not represent a complete loss of function phenotype. These cells were labeled at a time of depletion where a growth defect first became apparent, rather than when growth stopped. This was done in an attempt to define the immediate consequences of diminished Neo1p function. Likewise, neo1-ts cells were examined 1–3 h after temperature shift instead of the 6 h required to completely inhibit growth.
Surprisingly, neo1-ts cells accumulated a large number of small vesicles (30–50 nm) at the permissive temperature, where it is clear from the cell morphology and hypersensitivity to various chemicals that Neo1p is partially defective. This same vesicle accumulation phenotype is observed when sec21 is incubated at a semipermissive temperature (Rambourg et al., 1994). These vesicles are likely in transit between the ER and Golgi, but whether they are COPI or COPII vesicles is not known. Most of these vesicles disappear when neo1-ts is shifted to 37°C and ER and Golgi membranes accumulate. COPI mutants also accumulate ER at the nonpermissive temperature but the Golgi accumulation appears to be unique to neo1. This difference may reflect the hypomorphic nature of the neo1-ts alleles or that neo1 perturbs additional transport steps through, or from, the Golgi complex. However, it is important to note that neo1 does not appear to perturb COPII function and at least some proteins (Hsp150) are secreted normally. In addition, the CPY that escapes the ER-to-Golgi block in neo1 is sorted to the vacuole. Thus, a number of transport steps are occurring normally in neo1 and the defect appears rather specific to the Golgi-to-ER retrograde pathway.
Both COPI and neo1 mutations perturb glycosylation in the Golgi complex. For neo1-ts strains, the glycosylation defect appears to be the primary cause of the 37°C growth defect. This growth defect is sorbitol-remedial and the neo1-ts mutants are hypersensitive to calcofluor white. These phenotypes suggest an abnormal cell wall and are consistent with the Golgi-dependent glycosylation defects observed for CPY and -factor. How neo1 mutants perturb the function of Golgi glycosyltransferases is not known. However, localization of an -1,6-mannosyltransferase (Och1p) that initiates outer chain elongation is perturbed in sec21. Rather than being mislocalized to the vacuole, Och1p changes from a punctate distribution by immunofluorescence to a dispersed granular appearance that might reflect residence in small vesicles or Golgi fragments (Gaynor and Emr, 1997). In addition, two different -1,6-mannosyltransferase complexes containing Mnn9p, which elongate the outer chain on N-linked oligosaccharides, have been shown to cycle between the ER and Golgi complex. Like Rer1p, these proteins are mislocalized to the vacuole when retrograde transport is perturbed (Todorow et al., 2000). Therefore, it is possible that a reduced ability to form COPI vesicles in neo1 causes the glycosylation defect by mislocalizing -1,6-mannosyltransferases. The fact that neo1 lethality cannot be rescued by osmotic support indicates that Neo1p has an essential function independent of its role in glycosylation and cell wall synthesis. This essential Neo1p function appears to be in COPI-dependent Golgi-to-ER retrograde protein transport, an essential process in yeast.
How do potential APTs influence protein transport in the secretory pathway? Drs2p appears to play a direct role in forming secretory vesicles from Golgi membranes because inactivation of Drs2 or clathrin ts proteins causes a rapid loss of a specific class of exocytic vesicles (Gall et al., 2002). Drs2p also interacts directly with Gea2p, an ARF guanine nucleotide exchange factor, physically coupling Drs2p to vesicle budding machinery (Chantalat et al., 2004). The data presented in this work implicate Neo1p in COPI vesicle budding from early Golgi membranes, but whether this is a direct or indirect requirement remains to be determined. The onset of defects in transport is rather slow after shifting the neo1-ts mutants to the nonpermissive temperature. This could be explained by the hypomorphic nature of the neo1-ts alleles, but it could also indicate an indirect requirement for Neo1p in COPI vesicle formation. One model to explain the requirement for Neo1p is that an asymmetric distribution of phospholipids in the cis-Golgi is required for optimal COPI vesicle formation. ER and Golgi membranes in mammalian cells have been suggested to be asymmetric (Bollen and Higgins, 1980; Higgins, 1984). Neo1p might establish this phospholipid asymmetry, but it slowly decays after shifting the neo1-ts mutants to the nonpermissive temperature. As the membrane becomes more symmetric, the ability of ARF and COPI to form retrograde vesicles would diminish (Figure 12). It is also possible that Neo1p plays a more direct role in this process by interacting with components of the vesicle budding machinery, or by facilitating outward bending of the membrane during vesicle formation.
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We have recently found that late Golgi membranes purified from a drs2-ts mutant display a ts defect in translocating NBD-PS from the luminal to the cytosolic leaflet. The drs2 mutants also expose more PS on the outer leaflet of the plasma membrane than do wild-type cells (our unpublished results). These data strongly support the proposed APT activity for Drs2p. It is possible that both Neo1p and Drs2p control lipid asymmetry in the Golgi complex, with Neo1p acting in the cis compartments and Drs2p in the trans. In this case, loss of both proteins would severely perturb the transbilayer lipid distribution in the entire Golgi complex, perhaps explaining the synthetic lethality between drs2 and neo1-1.
COPI and clathrin-coated vesicles have been generated from synthetic liposomes using only cytosolic coat components (Spang and Schekman, 1998; Takei et al., 1998), suggesting that coat components alone are sufficient to drive vesicle formation without contribution from membrane proteins. However, these studies could not determine if vesicle formation occurred in vitro with the same rate and efficacy required in vivo. We have found that Neo1p, an essential P-type ATPase and integral membrane protein, is required for efficient Golgi-to-ER protein transport. All five members of the Drs2/Neo1 family proteins have now been implicated in protein trafficking at different steps in the secretory or endocytic pathways (Chen et al., 1999; Gall et al., 2002; Hua et al., 2002; Pomorski et al., 2003). The Drs2/Neo1 proteins appear to be APTs (Tang et al., 1996; Gomes et al., 2000; Pomorski et al., 2003), suggesting that the regulation of membrane phospholipid asymmetry plays an important role in the formation of coated transport vesicles.
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ACKNOWLEDGMENTS |
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* Corresponding author. E-mail address: tr.graham{at}vanderbilt.edu.
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REFERENCES |
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