Cytoplasm to vacuole trafficking of aminopeptidase I requires a t-SNARE-Sec1p complex composed of Tlg2p and Vps45p.
Aminopeptidase I (API) is imported into the yeast vacuole/lysosome by a constitutive non-classical vesicular transport mechanism, the cytoplasm to vacuole targeting (Cvt) pathway. Newly synthesized precursor API is sequestered in double-membrane cytoplasmic Cvt vesicles. The Cvt vesicles fuse with the vacuole, releasing single-membrane Cvt bodies containing proAPI into the vacuolar lumen, and maturation of API occurs when the Cvt body is degraded, releasing mature API. Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, non-selective mechanism that shares many similarities with the Cvt pathway. Here we show that Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway, but not in inducible macroautophagy. Fractionation and protease protection experiments indicate that Tlg2p is required prior to or at the step of API segregation into the Cvt vesicle. Thus, the early Vps45-Tlg2p-dependent step of the Cvt pathway appears to be mechanistically distinct from the comparable stage in macroautophagy. Vps45p associates with both the Tlg2p and Pep12p t-SNAREs, but API maturation is not blocked in a pep12(ts) mutant, indicating that Vps45p independently regulates the function of multiple t-SNARES at distinct trafficking steps.
Abeliovich
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UCSD School of Medicine, Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, 9500 Gilman Drive, La Jolla, CA 92093-0668, USA.
Darsow
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Emr
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D000626:Aminopeptidases / Q000378:metabolism*; D001343:Autophagy; D005656:Fungal Proteins / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008854:Microscopy, Electron; D051938:Munc18 Proteins; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D020123:Sirolimus / Q000494:pharmacology; D014617:Vacuoles / Q000378:metabolism; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011498:Protein Precursors; D050765:Qa-SNARE Proteins; C497931:SEC1 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C112252:TLG2 protein, S cerevisiae; C092934:VPS45 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D000626:Aminopeptidases; C019689:APE1 protein, S cerevisiae; D020123:Sirolimus