Novel targeting assay uncovers targeting information within peroxisomal ABC transporter Pxa1.

The mechanism behind peroxisomal membrane protein targeting is still poorly understood, with only two yeast proteins believed to be involved and no consensus targeting sequence. Pex19 is thought to bind peroxisomal membrane proteins in the cytosol, and is subsequently recruited by Pex3 at the peroxisomal surface, followed by protein insertion via a mechanism that is as-yet-unknown. However, some peroxisomal membrane proteins still correctly sort in the absence of Pex3 or Pex19, suggesting that multiple sorting pathways exist. Here, we studied sorting of yeast peroxisomal ABC transporter Pxa1. Co-localisation analysis of Pxa1-GFP in a collection of 86 peroxisome-related deletion strains revealed that Pxa1 sorting requires Pex3 and Pex19, while none of the other 84 proteins tested were essential. To identify regions with peroxisomal targeting information in Pxa1, we developed a novel in vivo re-targeting assay, using a reporter consisting of the mitochondrial ABC transporter Mdl1 lacking its N-terminal mitochondrial targeting signal. Using this assay, we showed that the N-terminal 95 residues of Pxa1 are sufficient for retargeting this reporter to peroxisomes. Interestingly, truncated Pxa1 lacking residues 1-95 still localised to peroxisomes. This was confirmed via localisation of various Pxa1 truncation and deletion constructs. However, localisation of Pxa1 lacking residues 1-95 depended on the presence of its interaction partner Pxa2, indicating that this truncated protein does not contain a true targeting signal.

Comparative Genomics of Peroxisome Biogenesis Proteins: Making Sense of the PEX Proteins.

PEX genes encode proteins involved in peroxisome biogenesis and proliferation. Using a comparative genomics approach, we clarify the evolutionary relationships between the 37 known PEX proteins in a representative set of eukaryotes, including all common model organisms, pathogenic unicellular eukaryotes and human. A large number of previously unknown PEX orthologs were identified. We analyzed all PEX proteins, their conservation and domain architecture and defined the core set of PEX proteins that is required to make a peroxisome. The molecular processes in peroxisome biogenesis in different organisms were put into context, showing that peroxisomes are not static organelles in eukaryotic evolution. Organisms that lack peroxisomes still contain a few PEX proteins, which probably play a role in alternative processes. Finally, the relationships between PEX proteins of two large families, the Pex11 and Pex23 families, were analyzed, thereby contributing to the understanding of their complicated and sometimes incorrect nomenclature. We provide an exhaustive overview of this important eukaryotic organelle.

The peroxisome biogenesis factors Pex3 and Pex19: multitasking proteins with disputed functions.

The peroxisomal membrane protein (PMP) Pex3 and its cytosolic interaction partner Pex19 have been implicated in peroxisomal membrane biogenesis. Although these peroxins have been extensively studied, no consensus has been reached yet on how they operate. Here, we discuss two major models of their function, namely, in direct insertion of proteins into the peroxisomal membrane or in formation of PMP-containing vesicles from the endoplasmic reticulum (ER). Pex3 can also recruit other proteins to the peroxisomal membrane (e.g., Inp1, Atg30, Atg36), thereby fulfilling roles in other processes such as autophagy and organelle retention. Recent studies indicate that Pex3 and Pex19 can also facilitate sorting of certain membrane proteins to other cellular organelles, including the ER, lipid droplets, and mitochondria.