CHARACTERIZATION OF YBR074 (PFF1), A CONSERVED VACUOLAR MEMBRANE METALLOPROTEASE FAMILY MEMBER

In yeast, the vacuole is the principal intracellular compartment associated with protein degradation. The vacuole acts as a buffering organelle that accumulates nutrients in times of plenty, and releases them into the cytosol during periods of nutrient starvation. This important biological function is mediated by vacuolar proteases, which exhibit a variety of conserved catalytic mechanisms. Metalloproteases represent one of the most diverse classes of proteases, and they are defined by a characteristic dependence on coordinated metal ions for their catalytic activity.\ud In higher eukaryotes, metalloproteases are associated with both intracellular homeostasis and remodeling of the extracellular environment. In humans, remodeling of the extracellular matrix is mediated by secreted matrix metalloproteases regulating cell motility during development and wound healing, and also serving as markers of cancer metastasis. Within the cell, metalloproteases play a major role in the maturation and trafficking of proteins, as well as in the turnover of long-lived, superfluous, or damaged proteins and organelles. \ud This dissertation represents the first characterization of the putative yeast metalloprotease Ybr074, which is named herein, protein in FXNA-related family (Pff1). Pff1 is a member of the conserved family of M28 metalloproteases, which includes the mammalian ortholog, FXNA. FXNA has been reported to be localized in the endoplasmic reticulum (ER), and is expressed in multiple tissues in rats, where it has been implicated in ovarian development. This dissertation shows that, unlike the ER-localized FXNA, Pff1 is a vacuolar protein. This finding is in agreement with extensive data, presented herein, demonstrating that Pff1 is not involved in protein quality control in the ER. However, genetic and chemical-genetic analyses suggest that Pff1 may have a role in yeast cell wall maintenance. Finally, this dissertation describes proteomic approaches employed in an attempt to identify endogenous substrates of Pff1, and outlines additional strategies aimed at defining the biological function of this novel vacuolar protease family member.\u