Identification of a cytoplasm to vacuole targeting determinant in aminopeptidase I.

Abstract

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...

Cvt18/Gsa12 Is Required for Cytoplasm-to-Vacuole Transport, Pexophagy, and Autophagy inSaccharomyces cerevisiaeandPichia pastoris

“…The intermediate band that appears following proteinase K digestion in the presence of detergent ( Figure 5B, top, lane 6) is thought to be due to an effect of detergent on the prApe1 conformation (Oda et al, 1996). From the above-mentioned observations, we concluded that Cvt18 is not required for the initial membrane association of prApe1, but it is involved in the membrane sequestration or enclosure stage of Cvt vesicle formation.…”

The machinery of macroautophagy

“…With regard to the details of cargo recognition, the Cvt pathway was the first well-studied type of selective autophagy in yeast; it is also the primary example of a biosynthetic rather than degradative use of autophagy [119][120][121][122]. The double-membrane sequestering vesicle is termed a Cvt vesicle rather than an autophagosome; as noted above, this vesicle contains selective cargos of the Cvt pathway, but excludes bulk cytoplasm, in accordance with the biosynthetic and constitutive nature of the Cvt pathway compared to starvation-induced autophagy.…”

Autophagic Processes in Yeast: Mechanism, Machinery and Regulation

“…During the initial identification of the genes encoding the Atg proteins, several were classified as being specific for nonselective macroautophagy, the Cvt pathway and/or pexophagy. For example, Atg11 was originally characterized as being a Cvt pathway-specific protein (Harding et al 1996;Oda et al 1996). These denotations, however, reflected the different screens used to identify the corresponding genes and the limited analyses available at that time.…”