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.
Kölling
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Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Hollenberg
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CP
D018528:ATP-Binding Cassette Transporters; D001692:Biological Transport / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012098:Reproduction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D043324:beta-Fructofuranosidase
D018528:ATP-Binding Cassette Transporters; D005656:Fungal Proteins; D006023:Glycoproteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase