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.

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.

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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.

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 α‐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 ΔDAKTI mutant was reduced compared with wild‐type Ste6, no ubiquitination could be detected for the more stable ΔA‐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.