Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

The vacuolar protein aminopeptidase I (API) uses a novel cytoplasm-to-vacuole targeting (Cvt) pathway. Complementation analysis of yeast mutants defective for cytoplasm-to-vacuole protein targeting (cvt) and autophagy (apg) revealed seven overlapping complementation groups between these two sets of mutants. In addition, all 14 apg complementation groups are defective in the delivery of API to the vacuole. Similarly, the majority of nonoverlapping cvt complementation groups appear to be at least partially defective in autophagy. Kinetic analyses of protein delivery rates indicate that autophagic protein uptake is induced by nitrogen starvation, whereas Cvt is a constitutive biosynthetic pathway. However, the machinery governing Cvt is affected by nitrogen starvation as targeting defects resulting from API overexpression can be rescued by induction of autophagy.The vacuole/lysosome is the major hydrolytic compartment of the cell and as such is central to survival during nitrogen starvation (1). When cells sense nitrogen starvation, cytoplasmic proteins and organelles are packaged nonselectively into autophagosomes, which are then targeted to the vacuole for degradation and turnover of their constituent components by resident hydrolases (2, 3). In this manner, amino acids as well as nucleic acids can be recycled to enable stress survival. In yeast, this autophagic process has been studied primarily through microscopic analysis. During autophagy, large doublemembraned vesicles (500 nm) nonselectively surround cytoplasmic proteins and organelles (2, 4). On delivery to the vacuole, the outermost membrane is proposed to fuse to the vacuolar membrane, resulting in the delivery of a still intact vesicle (autophagic body) into the vacuolar lumen (2, 5). These autophagic bodies are then degraded in a subsequent step that depends on proteinase B (4). Although two collections of autophagy mutants have been identified (6, 7), the molecular details of this process have not yet been elucidated.The resident hydrolases that mediate the digestive capacity of the vacuole are delivered to this organelle by two distinctly different protein transport pathways. The majority of vacuolar proteins are targeted through the secretory pathway (reviewed in refs. 8 and 9), whereas at least one vacuolar protein, aminopeptidase I (API) (10), uses the cytoplasm-to-vacuole targeting (Cvt) pathway. In mammalian cells, decreasing lumenal pH through the endocytic compartments is proposed to mediate the receptor/ligand dissociation reactions required for correct sorting through these compartments (11). Early evidence suggests that the same mechanism may mediate sorting in yeast as well; one important feature of the vacuolar membrane is the presence of a vacuolar ATPase (V-ATPase), which uses ATP to transport protons into the lumen. Maintenance of the resulting ApH is essential not only for optimal activity of many vacuolar proteases but also for efficient delivery of soluble vacuolar proteins by the secretory (12-14) and Cvt (15) pathways.The ...

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 ...

Abstract. Aminopeptidase I (API) is a soluble leucine aminopeptidase resident in the yeast vacuole (Frey, J., and K.H. Rohm. 1978. Biochim. Biophys. Acta. 527:31-41). The precursor form of API contains an amino-terminal 45-amino acid propeptide, which is removed by proteinase B (PrB) upon entry into the vacuole. The propeptide of API lacks a consensus signal sequence and it has been demonstrated that vacuolar localization of API is independent of the secretory pathway (Klionsky, D.J., R. Cueva, and D.S. Yaver. 1992. J. Cell Biol. 119:287-299). The predicted secondary structure for the API propeptide is composed of an amphipathic a-helix followed by a [3-turn and another a-helix, forming a helix-turn-helix structure. With the use of mutational analysis, we determined that the API propeptide is essential for proper transport into the vacuole. Deletion of the entire propeptide from the API molecule resuited in accumulation of a mature-sized protein in the cytosol. A more detailed examination using random mutagenesis and a series of smaller deletions throughout the propeptide revealed that API localization is severely affected by alterations within the predicted first a-helix. In vitro studies indicate that mutations in this predicted helix prevent productive binding interactions from taking place. In contrast, vacuolar import is relatively insensitive to alterations in the second predicted helix of the propeptide. Examination of API folding revealed that mutations that affect entry into the vacuole did not affect the structure of API. These data indicate that the API propeptide serves as a vacuolar targeting determinant at a critical step along the cytoplasm to vacuole targeting pathway.I n the yeast Saccharomyces cerevisiae soluble proteins enter the vacuole through one of four mechanisms: autophagy (Hirsch et al., 1992;Tsukada and Ohsumi, 1993;Thumm et al., 1994; reviewed in Seglen and Bohley, 1992), endocytosis (reviewed in Nothwehr and Stevens, 1994;Raths et al., 1993;Riezman, 1993), the secretory pathway (reviewed in Pryer et al., 1992), and the cytoplasm to vacuole targeting pathway (Cvt; Klionsky et al., 1992;Harding et al., 1995). In both autophagy and endocytosis there is bulk flow of soluble proteins into the vacuole but to date no soluble resident vacuolar protein has been shown to enter the yeast vacuole by these means.The majority of resident yeast vacuolar proteins enter this organelle through the secretory pathway (reviewed in Stack and Emr, 1993). Contrary to autophagy and endocytosis, proteins that transit through the secretory pathway are not engulfed by membranes but are translocated across the endoplasmic reticulum (ER) membrane. In a series of vesicle-mediated transport steps, the proteins are carried from the ER through the subcompartments of the Golgi before being directed to the vacuole (reviewed in Pryer et al., 1992 secretory pathway has been carefully documented for a number of vacuolar hydrolases (reviewed in Klionsky et al., 1990). These proteins translocate across the ER membrane via...

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.