Yol082p, a Novel CVT Protein Involved in the Selective Targeting of Aminopeptidase I to the Yeast Vacuole

Abstract

The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI).Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen. Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively. We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole. We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein. Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions. Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles. Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13-0.27-m round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082⌬C32p, or lacking Apg12p. In contrast to the autophagy mutants involved in API transport, a ⌬yol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway. The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers. Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19.In the yeast Saccharomyces cerevisiae the vacuolar hydrolase leucine aminopeptidase I (API) 1 is synthesized in the cytoplasm as a precursor (pAPI) (1) and delivered to the vacuole by one of two alternative routes that operate under distinct physiological conditions: the cytoplasm to vacuole targeting (Cvt), in nutrient-rich conditions, and the autophagy (Apg) pathway, under starvation conditions (2). The Cvt pathway is constitutive and biosynthetic, while autophagy is nonselective and degradative and is induced to survive periods of nutrient limitation (3). However, the two pathways share many molecular components and both involve sequestration by double-membrane saccular structures of unknown origin that capture the load, close into vesicles, and then fuse with the vacuole (4). A major difference between these pathways appears to be the size and content of the transport vesicles. The Cvt vesicles exclude cytoplasm and are smaller than autophagosomes that engulf bulk cytoplasm and even organelles (2). Strikingly, despite all these differences, targeting of API to the vacuole is specific and saturable, both in vegetative growth conditions and under nitrogen deprivation (3), although the molecular detail...

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