Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 Mutant.

Previous experiments suggested that trafficking of the a-factor transporter Ste6 of Saccharomyces cerevisiae to the yeast vacuole is regulated by ubiquitination. To define the ubiquitination-dependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation. We found that Ste6 accumulated at the vacuolar membrane in the doa4 mutant and not at the cell surface. Experiments with a doa4 pep4 double mutant showed that Ste6 uptake into the lumen of the vacuole is inhibited in the doa4 mutant. The uptake defect could be suppressed by expression of additional ubiquitin, indicating that it is primarily the result of a lowered ubiquitin level (and thus of reduced ubiquitination) and not the result of a deubiquitination defect. Based on our findings, we propose that ubiquitination of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway. In addition, we obtained evidence suggesting that Ste6 recycles between an internal compartment and the plasma membrane.

A family of small coiled-coil-forming proteins functioning at the late endosome in yeast.

The multispanning membrane protein Ste6, a member of the ABC-transporter family, is transported to the yeast vacuole for degradation. To identify functions involved in the intracellular trafficking of polytopic membrane proteins, we looked for functions that block Ste6 transport to the vacuole upon overproduction. In our screen, we identified several known vacuolar protein sorting (VPS) genes (SNF7/VPS32, VPS4, and VPS35) and a previously uncharacterized open reading frame, which we named MOS10 (more of Ste6). Sequence analysis showed that Mos10 is a member of a small family of coiled-coil-forming proteins, which includes Snf7 and Vps20. Deletion mutants of all three genes stabilize Ste6 and show a "class E vps phenotype." Maturation of the vacuolar hydrolase carboxypeptidase Y was affected in the mutants and the endocytic tracer FM4-64 and Ste6 accumulated in a dot or ring-like structure next to the vacuole. Differential centrifugation experiments demonstrated that about half of the hydrophilic proteins Mos10 and Vps20 was membrane associated. The intracellular distribution was further analyzed for Mos10. On sucrose gradients, membrane-associated Mos10 cofractionated with the endosomal t-SNARE Pep12, pointing to an endosomal localization of Mos10. The growth phenotypes of the mutants suggest that the "Snf7-family" members are involved in a cargo-specific event.

Functional asymmetry of the two nucleotide binding domains in the ABC transporter Ste6.

The yeast a-factor transporter Ste6 is a member of the ABC transporter family and is closely related to human MDR1. We constructed a set of 26 Ste6 mutants using a random mutagenesis approach. Cell fractionation experiments demonstrated that most of the mutants, with the notable exception of those with alterations in TM1, are transported to the plasma membrane, the presumptive site of action of Ste6. Trafficking, therefore, does not seem to be affected in most of the mutants. To identify regions in Ste6 that interact with the ABC transporter "signature motif" (LSGGQ) we screened for intragenic revertants of the LSGGQ mutant M68 (S507N). Suppressor mutations were identified in TM12 and upstream of TM6. Surprisingly, these mutations also suppressed the Walker A mutation G397D, which should be defective in ATP-binding and hydrolysis at NBD1. Photoaffinity labeling experiments with 8-azido-[alpha-32P]ATP showed that ATP binding at NBD2 is reduced by the suppressor mutation in TM12. The experiments further suggest that the two NBDs of Ste6 are not equivalent and affect each other's ability to bind and hydrolyze ATP.

The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport.

Leucine uptake by Saccharomyces cerevisiae is mediated by three transport systems, the general amino acid transport system (GAP), encoded by GAP1, and two group-specific systems (S1 and S2), which also transport isoleucine and valine. A new mutant defective in both group-specific transport activities was isolated by employing a gap1 leu4 strain and selecting for trifluoroleucine-resistant mutants which also showed greatly reduced ability to utilize L-leucine as sole nitrogen source and very low levels of [14C]L-leucine uptake. A multicopy plasmid containing a DNA fragment which complemented the leucine transport defect was isolated by selecting for transformants that grew normally on minimal medium containing leucine as nitrogen source and subsequently assaying [14C]L-leucine uptake. Transformation of one such mutant, lep1, restored sensitivity to trifluoroleucine. The complementing gene, designated LEP1, was subcloned and sequenced. The LEP1 ORF encodes a large protein that lacks characteristics of a transporter or permease (i.e., lacks hydrophobic domains necessary for membrane association). Instead, Lep1p is a very basic protein (pI of 9.2) that contains a putative bipartite signal sequence for targeting to the nucleus, suggesting that it might be a DNA-binding protein. A database search revealed that LEP1 encodes a polypeptide that is identical to Sac3p except for an N-terminal truncation. The original identification of SAC3 was based on the isolation of a mutant allele, sac3-1, that suppresses the temperature-sensitive growth defect of an actin mutant containing the allele act1-1. Sac3p has been previously shown to be localized in the nucleus. When a lep1 mutant was crossed with a sac3 deletion mutant, no complementation was observed, indicating that the two mutations are functionally allelic.

The linker region of the ABC-transporter Ste6 mediates ubiquitination and fast turnover of the protein.

Upon block of endocytosis, the a-factor transporter Ste6 accumulates in a ubiquitinated form at the plasma membrane. Here we show that the linker region, which connects the two homologous halves of Ste6, contains a signal which mediates ubiquitination and fast turnover of Ste6. This signal was also functional in the context of another plasma membrane protein. Deletion of an acidic stretch in the linker region ('A-box') strongly stabilized Ste6. The A-box contains a sequence motif ('DAKTI') which resembles the putative endocytosis signal of the alpha-factor receptor Ste2 ('DAKSS'). Deletion of the DAKTI sequence also stabilized Ste6 but, however, not as strongly as the A-box deletion. There was a correlation between the half-life of the mutants and the degree of ubiquitination: while ubiquitination of the deltaDAKTI mutant was reduced compared with wild-type Ste6, no ubiquitination could be detected for the more stable deltaA-box variant. Loss of ubiquitination seemed to affect Ste6 trafficking. In contrast to wild-type Ste6, which was associated mainly with internal membranes, the ubiquitination-deficient mutants accumulated at the plasma membrane, as demonstrated by immunofluorescence and cell fractionation experiments. These findings suggest that ubiquitination is required for efficient endocytosis of Ste6 from the plasma membrane.

Glove powder--a risk factor for the development of latex allergy?

Studies are described which compare the prevalence of sensitisation against latex proteins in medical personnel in different hospitals. The objective of these studies was to find out whether the use of powdered or unpowdered gloves could be related to the prevalence of latex allergy. Employees of one of the investigated hospitals (Germany) were using only powdered latex gloves, and in the other two hospitals (Great Britain) low protein powder-free latex gloves were used. Methods by which latex allergy can be avoided are suggested.

Characterization of the SAC3 gene of Saccharomyces cerevisiae.

A temperature-sensitive mutation (act1-1) in the essential actin gene of Saccharomyces cerevisiae can be suppressed by mutations in the SAC3 gene. A DNA fragment containing the SAC3 gene was sequenced. SAC3 codes for a 150 kDa hydrophillic protein which does not show any significant similarities with other proteins in the databases. Sac3 therefore is a novel yeast protein. A nuclear localization of Sac3 is suggested by the presence of a putative nuclear localization signal in the Sac3 sequence. A SAC3 disruption mutation was constructed. SAC3 disruption mutants were viable but grew more slowly and were larger than wild-type cells. In contrast to the sac3-1 mutation, the SAC3 disruption was not able to suppress the temperature sensitivity and the osmosensitivity of the act1-1 mutant. This demonstrates that act1-1 suppression by sac3-1 is not the result of a simple loss of SAC3 function. Furthermore, we examined the act1-1 and the sac3 mutants for defects in polarized cell growth by FITC-Concanavalin A (Con A)-labelling. The sac3 mutants showed a normal ConA-labelling pattern. In the act1-1 mutant, however, upon shift to non-permissive temperature, newly synthesized cell wall material, instead of being directed towards the bud, was deposited at discrete spots in the mother cell.

The hemolysin B protein, expressed in Saccharomyces cerevisiae, accumulates in binding-protein (BiP)-containing structures.

The hemolysin B (Hly B) protein of Escherichia coli, a member of the ABC-transporter family, was expressed in Saccharomyces cerevisiae and tested for its ability to complement a defect in the a-factor transporter Ste6. We found that HlyB was not able to restore mating ability to a STE6 deletion strain. The HlyB protein did not co-fractionate with Ste6 on sucrose gradients, indicating that improper localization of the HlyB protein could contribute to the lack of complementation. Immunofluorescence experiments suggest that HlyB is localized to structures derived from the endoplasmic reticulum (ER). The HlyB-expressing cells revealed a perinuclear staining typical of ER-localized proteins and intensely staining ring-like structures (HlyB-bodies). Double-label immunofluorescence experiments show that the HlyB structures also contain the ER binding protein (BiP), the product of the KAR2 gene. The HlyB protein, however, did not co-fractionate with another ER marker protein, the NADPH cytochrome c reductase. The HlyB bodies could be derivatives of a novel compartment of the early secretory pathway which contains BiP but not other resident ER proteins. In this case, HlyB could serve as a tool for the biochemical characterization of this compartment.

The SAC3 gene encodes a nuclear protein required for normal progression of mitosis.

The SAC3 gene of Saccharomyces serevisiae has been implicated in actin function by genetic experiments showing that a temperature sensitive mutation in the essential actin gene (actl-1) can be suppressed by mutations in SAC3. An involvement of SAC3 in actin function is further suggested by the observation that the actin cytoskeleton is altered in SAC3 mutants. Our fractionation experiments, however, point to a nuclear localization of Sac3p. On sucrose density gradients Sac3p co-fractionated with the nuclear organelle markers examined. Furthermore, Sac3p was enriched 10-fold in a nuclei preparation along with the nuclear protein Nop1p. In this report we further show that SAC3 function is required for normal progression of mitosis. SAC3 mutants showed a higher fraction of large-budded cells in culture, indicative of a cell cycle delay. The predominant population among the large-budded sac3 cells were cells with a single nucleus at the bud-neck and a short intranuclear spindle. This suggests that a cell cycle delay occurs in mitosis prior to anaphase. The observation that SAC3 mutants lose chromosomes with higher frequency than wild-type is another indication for a mitotic defect in SAC3 mutants. We further noticed that SAC3 mutants are more resistant against the microtubule destabilizing drug benomyl. This finding suggests that SAC3 is involved, directly or indirectly, in microtubule function. In summary, our data indicate that SAC3 is involved in a process which affects both the actin cytoskeleton and mitosis.

The first hydrophobic segment of the ABC-transporter, Ste6, functions as a signal sequence.

The Ste6 protein of Saccharomyces cerevisiae is a member of the ABC-transporter family containing 12 putative membrane spanning segments. To test whether Ste6 is inserted into the endoplasmic reticulum (ER) membrane by a sequential insertion mechanism we constructed a Ste6-invertase fusion containing the first hydrophobic segment of Ste6 fused to invertase lacking its own signal sequence. The resulting protein became glycosylated demonstrating that it was translocated across the ER-membrane. The finding that the N-terminal hydrophobic segment of Ste6 is recognized by the ER-translocation machinery suggests that Ste6 is inserted sequentially into the ER-membrane. Furthermore, our experiments support the Nin orientation of Ste6 predicted from the Ste6 sequence. Several findings suggest that invertase is cleaved from the Ste6 membrane anchor: (i) the gel mobility of deglycosylated wild-type invertase and fusion protein derived invertase is the same; (ii) the periplasmic invertase activity is found in the cell wall fraction, i.e. it is not associated with the cell body; (iii) a signal peptide cleavage site is predicted in the Ste6 sequence. Although the membrane anchor appeared to be cleaved, most of the invertase was retained in the ER, probably due to aggregate formation.

Nucleotide sequence of the SAC2 gene of Saccharomyces cerevisiae.

A temperature-sensitive mutation (act1-1) in the essential actin gene of Saccharomyces cerevisiae can be suppressed by mutations in the SAC2 gene. A cloned genomic DNA fragment that complements the cold-sensitive growth phenotype associated with such a suppressor mutation (sac2-1) was sequenced. The fragment contained an open reading frame that encodes a 641 amino acid predicted hydrophilic protein with a molecular weight of 74,445. No sequences with significant similarity to SAC2 were found in the GenBank and EMBL databases. A SAC2 disruption mutation was constructed which had phenotypes similar to the sac2-1 point mutation. A haploid SAC2 disruption strain failed to grow at low temperature and the disruption allele suppressed the temperature-sensitive act1-1 growth defect. The suppression phenotype was dependent on the strain background.

The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants.

We are investigating the transport and turnover of the multispanning membrane protein Ste6. The Ste6 protein is a member of the ABC-transporter family and is required for the secretion of the yeast mating pheromone a-factor. In contrast to the prevailing view that Ste6 is a plasma membrane protein, we found that Ste6 is mainly associated with internal membranes and not with the cell surface. Fractionation and immunofluorescence data are compatible with a Golgi localization of Ste6. Despite its mostly intracellular localization, the Ste6 protein is in contact with the cell surface, as demonstrated by the finding that Ste6 accumulates in the plasma membrane in endocytosis mutants. The Ste6 protein which accumulates in the plasma membrane in endocytosis mutants is ubiquitinated. Ste6 is thus the second protein in yeast besides MAT alpha 2 for which ubiquitination has been demonstrated. Ste6 is a very unstable protein (half-life 13 min) which is stabilized approximately 3-fold in a ubc4 ubc5 mutant, implicating the ubiquitin system in the degradation of Ste6. The strongest stabilizing effect on Ste6 is, however, observed in the vacuolar pep4 mutant (half-life > 2 h), suggesting that most of Ste6 is degraded in the vacuole. Secretory functions are required for efficient degradation of Ste6, indicating that Ste6 enters the secretory pathway and is transported to the vacuole by vesicular carriers.

Synthesis and farnesylation of a-factor fusion proteins in Saccharomyces cerevisiae.

We have generated in-frame fusions between the mouse dihydrofolate reductase (DHFR) and parts of the a-factor MFA1 gene to explore the potential of a-factor as a secretion signal for larger polypeptides. We demonstrated that the fusion proteins are farnesylated by comparing the mobility of fusion proteins prepared from a wild-type strain and a farnesyltransferase mutant (ste16/ram1) on SDS-gels and by an in vitro farneyslation assay. In contrast to unmodified DHFR, the fusion proteins could be sedimented from cell extracts by centrifugation. Solubilization experiments indicated that the highly hydrophobic a-factor moiety renders the fusion proteins insoluble, explaining why the fusions are not secreted into the culture medium.

A new yeast gene with a myosin-like heptad repeat structure.

We isolated a gene encoding a 218 kDa myosin-like protein from Saccharomyces cerevisiae using a monoclonal antibody directed against human platelet myosin as a probe. The protein sequence encoded by the MLP1 gene (for myosin-like protein) contains extensive stretches of a heptad-repeat pattern suggesting that the protein can form coiled coils typical of myosins. Immunolocalization experiments using affinity-purified antibodies raised against a TrpE-MLP1 fusion protein showed a dot-like structure adjacent to the nucleus in yeast cells bearing the MLP1 gene on a multicopy plasmid. In mouse epithelial cells the yeast anti-MLP1 antibodies stained the nucleus. Mutants bearing disruptions of the MLP1 gene were viable, but more sensitive to ultraviolet light than wild-type strains, suggesting an involvement of MLP1 in DNA repair. The MLP1 gene was mapped to chromosome 11, 25 cM from met1.

Transcription in the region of the replication origin, oriC, of Escherichia coli: termination of asnC transcripts.

Transcription from the asnC promoter was found to proceed through the replication origin, oriC, into the gidA gene of Escherichia coli. Between 10% and 20% of asnC transcripts reached oriC in vivo. Termination sites in the intergenic region between asnC and mioC and within mioC were determined in vivo and in vitro using S1 mapping. Only about 10% of the transcripts terminated at the asnC terminator in vivo. DnaA protein dependent termination was observed close to the binding site, dnaA box, for DnaA protein. In the in vitro replication system asnC transcripts did not reach oriC, suggesting that asnC transcripts are not involved in the initiation of replication, contrary to mioC transcripts. We suggest that oriC and mioC might have been transposed during evolution into an asnC regulation.

AsnC, a multifunctional regulator of genes located around the replication origin of Escherichia coli, oriC.

The expression of the gidA gene which is located immediately counterclockwise of the replication origin of Escherichia coli, oriC, was found to be negatively regulated by the AsnC protein in an in vitro transcription-translation system. This effect is not due to simple repression of transcription originating at the gidA promoter, because the AsnC protein did not change the level of gidA promoter dependent transcription as analysed by promoter-galK fusions and by S1 mapping. From these data we conclude that the AsnC protein controls gidA gene expression at a post-transcriptional level. gidA is the third gene in the oriC region, besides asnA and asnC, whose expression is under AsnC control. However, the mechanisms involved are different: regulation of transcription in the case of asnA and asnC and post-transcriptional control of gidA. The gidA promoter was mapped by deletion analysis and by S1 mapping. We defined two regions that affect promoter activity negatively. Additional transcripts, regulated by AsnC, started more than 300 bp upstream of the gidA promoter and were found to enter the gidA region. These transcripts, originating either at the mioC and/or the ansC promoter traverse the replication origin.

Transcripts within the replication origin, oriC, of Escherichia coli.

Transcription start and termination sites were mapped in the E. coli replication origin, oriC. Outward transcription from within oriC (promoters Pori-r and Pori-l) was found to start in vivo at position 178 for Pori-l and at positions 294 and 304 for Pori-r, respectively. These transcripts were terminated after 100-150 bases, at terminators designated Tori-l and Tori-r. Transcription from the 16 kd promoter, which lies clockwise adjacent to oriC and promotes transcription toward oriC, started at position 757 and gave transcripts with 3' ends at several positions within and to the left of the minimal replication origin. However, the majority of transcripts traversed the whole oriC region, and were not terminated within the DNA segment tested. Transcription of the chromosomal 16 kd gene was negatively regulated by DnaA protein and positively affected by dam methylation. The possible function of these transcripts is discussed.

Regulation of transcription of the chromosomal dnaA gene of Escherichia coli.

By comparative S1 analysis we investigated the in vivo regulation of transcription of the chromosomal dnaA gene coding for a protein essential for the initiation of replication at the chromosomal origin. Inactivation of the protein in dnaA mutants results in derepression, whereas excess DnaA protein (presence of a DnaA overproducing plasmid) leads to repression of dnaA transcription. Both dnaA promoters are subject to autoregulation allowing modulation of transcriptional efficiency by at least 20-fold. Increasing the number of oriC sequences (number of DnaA binding sites) in the cell by introducing oriC plasmids leads to a derepression of transcription. Autoregulation and binding to oriC suggest that the DnaA protein exerts a major role in the regulation of the frequency of initiation at oriC. The efficiency of transcription of the dnaA2 promoter is reduced in the absence of dam methylation, which is involved in the regulation of oriC replication.

AsnC: an autogenously regulated activator of asparagine synthetase A transcription in Escherichia coli.

The regulation of the asparagine synthetase A gene of Escherichia coli was studied in vitro with a coupled transcription-translation system. It was shown that the 17-kilodalton gene, which is transcribed divergently from the adjacent asnA gene, codes for an activator of asnA transcription. The synthesis of the 17-kilodalton protein, which we now call AsnC, is autogenously regulated. The stimulating effect of AsnC on asnA transcription is abolished by asparagine, while the autoregulation of asnC is not affected by asparagine. The N-terminal part of the asnC protein, inferred from the DNA sequence, is homologous to the DNA-binding domain of regulatory proteins like catabolite gene activator, cro, and cI. This homology and direct repeats found in the region of the two asn promoters suggest that the asnC protein regulates transcription by binding to DNA. The asn promoters were defined by mapping of the mRNA start sites of in vitro-generated transcripts.

dnaA protein-regulated transcription: effects on the in vitro replication of Escherichia coli minichromosomes.

Both initiation of replication and initiation of transcription are influenced by dnaA protein, when minichromosomes are assayed in vitro for dnaA protein complementation. This dnaA protein effect is seen only if minichromosomes are used containing the 16-kd promoter, from which transcription is directed into the minimal origin. Determination of the 16-kd promoter activity both in vivo and in vitro showed that this strong promoter is specifically repressed by dnaA protein. The 16-kd promoter is thus an integral regulatory region of oriC.