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 con stitutive non-classical vesicular transport mechanism, the cytoplasm to vac uole targeting (Cvt) pathway. Newly synthesized precursor API is sequestere d in double-membrane cytoplasmic Cvt vesicles. The Cvt vesicles fuse with t he vacuole, releasing single-membrane Cvt bodies containing pro-API into th e vacuolar lumen, and maturation of API occurs when the Cvt body is degrade d, releasing mature API, Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, nonselective mechanism that s hares many similarities with the Cvt pathway. Here we show that Tlg2p, a me mber of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homolo gue, are required in the constitutive Cvt pathway, but not in inducible mac roautophagy, Fractionation and protease protection experiments indicate tha t 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 app ears to be mechanistically distinct from the comparable stage in macroautop hagy, Vps45p associates with both the Tlg2p and Pep12p t-SNAREs, but API ma turation is not blocked in a pep12(ts) mutant, indicating that Vps45p indep endently regulates the function of multiple t-SNARES at distinct traffickin g steps.