CHARACTERIZATION OF YBR074 (PFF1), A CONSERVED VACUOLAR MEMBRANE METALLOPROTEASE FAMILY MEMBER by Karen Alice Hecht H.B.Sc. Biochemistry, University of Toronto, Toronto Canada, 2006 Submitted to the Graduate Faculty of The Kenneth P. Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2013 UNIVERSITY OF PITTSBURGH FACULTY OF DIETRICH SCHOOL OF ARTS AND SCIENCES This dissertation was presented by Karen Alice Hecht It was defended on July 27, 2012 and approved by Meir Aridor, Associate Professor, Cell Biology and Physiology Karen M. Arndt, Professor, Biological Sciences Michael Grabe, Assistant Professor, Biological Sciences Joseph A. Martens, Assistant Professor, Biological Sciences Committee chair: Jeffrey L. Brodsky, Professor, Biological Sciences ii Copyright © by Karen Alice Hecht 2013 iii CHARACTERIZATION OF YBR074 (PFF1), A CONSERVED VACUOLAR MEMBRANE METALLOPROTEASE FAMILY MEMBER Karen Alice Hecht, PhD University of Pittsburgh, 2013 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. 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. 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 iv 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. v TABLE OF CONTENTS PREFACE ................................................................................................................................. XIV 1.0 INTRODUCTION ........................................................................................................ 1 1.1 STRUCTURE AND FUNCTION OF THE YEAST VACUOLE ................... 2 1.1.1 Proteolysis......................................................................................................... 8 1.1.1.1 Autophagy .............................................................................................. 8 1.1.1.2 Microautophagy .................................................................................. 13 1.1.1.3 Endocytosis .......................................................................................... 13 1.1.2 Trehalose metabolism.................................................................................... 17 1.1.3 Other functions of the vacuole ...................................................................... 18 1.2 VACUOLAR PROTEASES ............................................................................. 20 1.2.1 Soluble vacuolar proteases. ........................................................................... 21 1.2.2 Membrane-bound vacuolar protease ........................................................... 30 1.3 METALLOPROTEASE FAMILY MEMBERS IN YEAST ......................... 31 1.4 QUESTIONS FOR FUTURE RESEARCH ON YEAST VACUOLAR FUNCTION ......................................................................................................................... 35 1.5 DISSERTATION GOALS AND OVERVIEW ............................................... 36 2.0 CHARACTERIZATION AND LOCALIZATION OF YBR074 .......................... 41 2.1 INTRODUCTION ............................................................................................. 41 vi 2.2 MATERIALS AND METHODS ...................................................................... 42 2.2.1 Computational Methods................................................................................ 42 2.2.2 Strains, plasmids, yeast growth conditions, and molecular techniques .... 43 2.2.3 Protein localization ........................................................................................ 49 2.2.4 Cycloheximide chase analysis of Ybr074 stability ...................................... 52 2.3 RESULTS ........................................................................................................... 53 2.4 CONCLUSIONS ................................................................................................ 70 3.0 ATTEMPTS TO DEFINE THE FUNCTION OF YBR074 ................................... 71 3.1 INTRODUCTION ............................................................................................. 71 3.2 MATERIALS AND METHODS ...................................................................... 72 3.2.1 Strains ............................................................................................................. 72 3.2.1.1 Generating double mutant strains by yeast mating ......................... 73 3.2.2 Growth Assays ............................................................................................... 73 3.2.3 Molecular Techniques ................................................................................... 75 3.2.4 Assays examining ER-Associated Degradation (ERAD) ........................... 75 3.2.4.1 Cycloheximide chase analyses ............................................................ 75 3.2.4.2 Cycloheximide pulse chase analyses .................................................. 78 3.2.5 Analysis of phylogenetic profiling data ....................................................... 79 3.2.6 Proteomic analysis methods .......................................................................... 79 3.3 RESULTS ........................................................................................................... 81 3.3.1 Is Ybr074 a quality control protease?.......................................................... 81 3.3.1.1 Does Ybr074 function as a general protease during ERAD? .......... 82 vii 3.3.1.2 Does Ybr074 help resolve ERAD substrates with large lumenal domains? ............................................................................................................. 87 3.3.1.3 Do Ybr074 and the 26S proteasome function in synergy during the degradation of CFTR? ...................................................................................... 90 3.3.1.4 Does ablation of YBR074 and 26S proteasome function lead to synergistic effects on cell growth? .................................................................... 92 3.3.2 Do genetic and chemical-genetic interactions between YBR074 and other genes link this protein to cellular processes? ........................................................... 95 3.3.2.1 Does YBR074 play a role in yeast mating? ...................................... 100 3.3.2.2 Does YBR074 function in the transition to pseudohyphal growth in nutrient-limited conditions? ........................................................................... 101 3.3.3 Does the phylogenetic profile of YBR074 hint at its function? ................ 103 3.3.4 Can a substrate for Ybr074 be identified using proteomic approaches? 106 3.4 CONCLUSIONS .............................................................................................. 120 4.0 CELL WALL INTEGRITY .................................................................................... 122 4.1 MATERIALS AND METHODS .................................................................... 124 4.1.1 Strains and Molecular Techniques ............................................................ 124 4.1.2 Zymolyase sensitivity Assay ........................................................................ 124 4.1.3 β-1,3-glucan quantification assay ............................................................... 124 4.1.4 Killer toxin halo assay ................................................................................. 125 4.2 RESULTS ......................................................................................................... 126 4.2.1 Does YBR074 interact with the cell wall integrity function of CDC48? .. 129 4.2.2 Does the loss of YBR074 affect the