Abstract. In Saccharomyces cerevisiae the vacuolar protein aminopeptidase I (API) is localized to the vacuole independent of the secretory pathway. The alternate targeting mechanism used by this protein has not been characterized. API is synthesized as a 61-kD soluble cytosolic precursor. Upon delivery to the vacuole, the amino-terminal propeptide is removed by proteinase B (PrB) to yield the mature 50-kD hydrolase. We exploited this delivery-dependent maturation event in a mutant screen to identify genes whose products are involved in API targeting. Using antiserum to the API propeptide, we isolated mutants that accumulate precursor API. These mutants, designated cvt, fall into eight complementation groups, five of which define novel genes. These five complementation groups exhibit a specific defect in maturation of API, but do not have a significant effect on vacuolar protein targeting through the secretory pathway. Localization studies show that precursor API accumulates outside of the vacuole in all five groups, indicating that they are blocked in API targeting and/or translocation. Future analysis of these gene products will provide information about the subcellular components involved in this alternate mechanism of vacuolar protein localization.

We have explored the phenotypic and genetic overlap between autophagocytosis and cytoplasm to vacuole targeting in the yeast Saccharomyces cerevisiae.Complementation analysis was performed with mutants in each of these groups (aut and cvt, respectively), and three complementation groups were found to overlap. Also, most of the unique aut mutants accumulated precursor aminopeptidase I in the cytoplasm, while maintaining wild type kinetics and maturation of proteins targeted to the vacuole via the secretory pathway. The majority of the non-overlapping cvt mutants were found to be at least partially defective in autophagy. Some mutants in each group, however, appear to be only marginally affected in the other phenotype, implying that these pathways only partially overlap. We propose that import of aminopeptidase I into the vacuole shares a number of components required for bulk autophagocytosis, but is made specific, saturable, and constitutive by the presence of a receptor or other interacting protein(s).Until recently it was thought that all resident proteins reach the vacuole through a portion of the secretory pathway, transiting from the endoplasmic reticulum (ER) 1 to the Golgi complex before being sorted into the vacuolar delivery pathway. In Saccharomyces cerevisiae, however, the vacuolar proteins aminopeptidase I (API) and ␣-mannosidase are targeted to the vacuole directly from the cytoplasm, independent of the secretory pathway (1, 2). The alternate targeting mechanism used by API has been partially characterized. API is synthesized as a soluble cytosolic protein containing an amino-terminal propeptide. Upon delivery to the vacuole, this propeptide is proteolytically removed in a proteinase B (PrB)-dependent manner, to generate the mature form of the enzyme. The signal used by API that allows it to be directed to the vacuole has been localized to a putative ␣ helix at the very amino terminus of the propeptide (3). Overproduction of API results in accumulation of the precursor form of this hydrolase, suggesting that one or more of the component(s) required for its import may be saturable (1). Precursor accumulation might result from the titration of either cytosolic or membrane proteins required for API import. Recently, we have isolated a group of unique mutants that block the targeting of API to the yeast vacuole (4).The actual mechanism of protein import employed in the targeting of API is still open to question, but two distinct mechanisms are possible: entry through a proteinaceous pore or via a vesicle-mediated process. The observation that API import is inhibited at 14°C (5) argues against direct translocation through a protein channel. Also, precursor API in the cytosol appears to be folded, as indicated by protease resistance (3), rather than in an extended form. There are two known vesicle-mediated pathways, endocytosis and autophagy, used for degradative delivery of proteins to the vacuole/lysosome from the plasma membrane/cell surface or cytoplasm, respectively. In mammalian cells, autophagy is ...

In Saccharomyces cerevisiae the vacuoles are partitioned from mother cells to daughter cells in a cell-cycle-coordinated process. The molecular basis of this event remains obscure. To date, few yeast mutants had been identified that are defective in vacuole partitioning (vac), and most such mutants are also defective in vacuole protein sorting (vps) from the Golgi to the vacuole. Both the vps mutants and previously identified non-vps vac mutants display an altered vacuolar morphology. Here, we report a new method to monitor vacuole inheritance and the isolation of six new non-vps vac mutants. They define five complementation groups (VAC8-VAC12). Unlike mutants identified previously, three of the complementation groups exhibit normal vacuolar morphology. Zygote studies revealed that these vac mutants are also defective in intervacuole communication. Although at least four pathways of protein delivery to the vacuole are known, only the Vps pathway seems to significantly overlap with vacuole partitioning. Mutants defective in both vacuole partitioning and endocytosis or vacuole partitioning and autophagy were not observed. However, one of the new vac mutants was additionally defective in direct protein transport from the cytoplasm to the vacuole.

The accumulation of trehalose is a critical determinant of stress resistance in the yeast Saccharomyces cerevisiae. We have constructed a yeast strain in which the activity of the trehalose-hydrolyzing enzyme, acid trehalase (ATH), has been abolished. Loss of ATH activity was accomplished by disrupting the ATH1 gene, which is essential for ATH activity. The ⌬ath1 strain accumulated greater levels of cellular trehalose and grew to a higher cell density than the isogenic wild-type strain. In addition, the elevated levels of trehalose in the ⌬ath1 strain correlated with increased tolerance to dehydration, freezing, and toxic levels of ethanol. The improved resistance to stress conditions exhibited by the ⌬ath1 strain may make this strain useful in commercial applications, including baking and brewing.