Signals, receptors, and protein targeting: Determining the molecular mechanisms for peroxisome biogenesis in higher plants.

Abstract

Peroxisomes are essential organelles found in almost all eukaryotic cells. Improperly formed peroxisomes result in numerous fatal metabolic disorders in humans. New peroxisomes are formed by the fission of preexisting peroxisomes and posttranslational import of peroxisomal. matrix enzymes. The process of peroxisome biogenesis in plants is studied using radiolabeled peroxisomal proteins and isolated peroxisomes in an <italic>in vitro</italic> import assay. To understand the process of peroxisome biogenesis I have analyzed the targeting determinants, energy requirements, and molecular mechanisms of receptor-mediated protein import. I have determined that protein import into isolated peroxisomes requires a carboxyl-terminal targeting signal known as the Peroxisomal Targeting Signal type 1 (PTS1). The carboxyl-terminal targeting determinant is comprised of three amino acids (Serine-Arginine-Leucine) and is recognized by Pex5p, the PTS1 receptor. I have analyzed the role of the PTS1 receptor biochemically using the <italic>in vitro</italic> import assay. When added to standard import reactions, Pex5p stimulates the amount of protein that is imported into organelles, revealing its role as a soluble receptor. I have shown that Pex5p escorts its protein cargo into the peroxisomal matrix, which is consistent with the extended shuttle mechanism for PTS1 protein import. In addition, the import process requires the hydrolysis of nucleotide triphosphates (NTPs) as well as components of a protonmotive force (PMF). The energy released during NTP hydrolysis could be used by chaperones, ATPases, or protein translocases during the import process. The role of a PMF in posttranslational import of PTS1 proteins was further examined. I determined that PTS1 protein import is facilitated by the low pH environment of the peroxisomal matrix. Immunoprecipitation experiments using a range of buffer pH revealed that the interaction between receptor and ligand is pH-dependent. I demonstrated that Pex5p and PTS1 proteins interact well at a cytosolic pH (pH 7) and less well at the proposed matrix pH (pH 6), thus providing a possible mechanistic model for the net accumulation of PTS1 proteins in the peroxisomal matrix during peroxisome biogenesis. The reconstitution of peroxisomal protein import allows us to characterize the cellular requirements and targeting mechanisms for peroxisome biogenesis.