Biochemical Analysis of the Regulation of Vesicle Fusion, Peroxisomal Protein Import and Proteolytic Processing in Eukaryotes.

[摘要] My dissertation research provides major contributions to the current understanding of intracellular protein trafficking in two important areas: vesicular fusion and peroxisome biogenesis. I began with a study of SNAREs, highly conserved proteins that form the core fusion machinery within secretory and endosomal trafficking systems. I examined the regulation of an integral member of this protein machinery, syntaxin 5, by its mammalian endoplasmic reticulum/Golgi Sec1/Munc18 protein binding partner rSly1. To address their functional relationship, I produced a conformation-specific monoclonal antibody to use in immunostaining experiments and in vitro ER-to-Golgi transport assays. Results from the manipulation of rSly1/syntaxin 5 interactions indicate that rSly1 function is intimately associated with syntaxin binding, not promoting availability of the SNARE motif, but perhaps supporting a later step in SNARE complex formation.My research continued with an examination of peroxisome matrix protein import, a mode of protein transport that utilizes two distinct peroxisomal targeting signals (PTS1 and 2) to bind exclusively to two different cytosolic receptors that define separate import pathways. A few matrix enzymes contain a PTS2 that is cleaved off after import, in plants and mammals. The identity of the PTS2 processing protease is unknown. Bioinformatic analysis of the Arabidopsis genome revealed a 76kDa Deg-protease, AtDEG15, predicted to be peroxisomal. Analysis of protein extracts from Arabidopsis plants with a knockout mutation in atdeg15 reveals that the precursor form of the PTS2 protein thiolase (THL) was not processed. Using reverse transcription, I isolated and cloned AtDEG15. In vitro peroxisome import, protease assays, and mutagenic analysis demonstrated that AtDEG15 is a novel PTS2-specific processing protease.Two proteins found in Arabidopsis thaliana have been identified that possess both a putative PTS1 and a PTS2, long-chain acyl-CoA synthetase 7 (LACS7) and alpha-crystallin domain protein 31.2 (ACD31.2). While both proteins have been localized to the peroxisome, the PTS responsible for their localization remains unclear. Mutagenic analysis and standard in vitro import assays showed that either the PTS1 or PTS2 is sufficient to direct ACD31.2 and LACS7 import.Thus, using cellular and molecular biological techniques, my research has expanded the current understanding of intracellular protein trafficking.

[发布机构] University of Michigan

[效力级别] Peroxisomal Processing Protease

[关键词] SM Protein Regulation;Peroxisomal Processing Protease;Dual-signal Protein Targeting;Molecular;Cellular and Developmental Biology;Science;Cellular & Molecular Biology