A new class of mutants deficient in dodecamerization of aminopeptidase 1 and vacuolar transport

Vacuolar aminopeptidase 1 is transported to the vacuole by cytoplasmic doub le-membrane vesicles, the nonclassic Cvt pathway The cytosolic protein dode camerizes and is enclosed in a double-membrane vesicle, which is transporte d to and fuses with the vacuole releasing a single-membrane autophagic body into the vacuolar lumen. This is degraded and the precursor sequence of am inopeptidase 1 is removed. This pathway resembles autophagy, and most prote ins identified to function in the Cvt pathway are also required for autopha gy and vice versa. The cytosolic precursor protein and the matured vacuolar protein form a homododecameric complex, and only this complex has enzymati c activity. We developed a new genetic screen to isolate mutants in the bio genesis of vacuolar aminopeptidase 1 based on its enzymatic activity. The s ensitivity of this assay made it possible for us to search for mutants unde r conditions where autophagy is down-regulated, and me describe two new mut ants defective in the biogenesis pathway of vacuolar aminopeptidase 1. Muta nts are defective in dodecamerization of pApe1p and in Cvt vesicle formatio n. Complex assembly and transport vesicle formation appear to be linked pro cesses. This mechanism can control the potentially harmful cytoplasmic prot eolytic activity and could be the driving force for this nonclassic mechani sm of vacuolar enzyme transport.