The yeast endosomal t-SNARE, Pep12p, functions in the absence of its transmembrane domain.
Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of two distinct SNARE complexes. Pep12p and Vam3p are both t-SNAREs of the syntaxin family that are components of these SNARE complexes. We have used a genetic approach to address the role of Pep12p in vacuolar protein transport. Our screen for temperature-sensitive pep12 mutants yielded six alleles that were rapidly inactivated upon exposure to the non-permissive temperature. Surprisingly, the proteins encoded by these alleles were all truncated immediately prior to the transmembrane domain. Here we demonstrate that Pep12p requires its transmembrane domain for proper localization, but not for its role in vesicle fusion. In addition, we show that although Pep12p can replace Vam3p in the vacuolar SNARE complex, its transmembrane domain is required to function at this step. Therefore, the transmembrane domain of Pep12p performs different roles in the prevacuolar and vacuolar SNARE complexes.
Gerrard
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SR
Institute of Molecular Biology, Department of Chemistry, University of Oregon, Eugene, OR 97403-1229, USA.
Mecklem
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AB
Stevens
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TH
D000595:Amino Acid Sequence; D002458:Cell Fractionation; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D013696:Temperature; D014170:Transformation, Genetic; D022161:Transport Vesicles / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D008565:Membrane Proteins; C081224:PEP12 protein, S cerevisiae; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C107706:VAM3 protein, S cerevisiae; D033921:Vesicular Transport Proteins