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.
Kölling
R
R
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany. ralf.koelling@uni-duesseldorf.de
Losko
S
S
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins; D006207:Half-Life; D007527:Isoenzymes / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D005656:Fungal Proteins; D006023:Glycoproteins; D007527:Isoenzymes; D009097:Multienzyme Complexes; C434364:PMA2 protein, S cerevisiae; D021382:Protein Sorting Signals; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014452:Ubiquitins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases