Pex19p binds Pex30p and Pex32p at regions required for their peroxisomal localization but separate from their peroxisomal targeting signals
ABSTRACT The assembly of proteins in the peroxisomal membrane is a multistep process requiring their recognition in the cytosol, targeting to and insertion into the peroxisomal membrane, and stabilization within the lipid bilayer. The peroxin Pex19p has been proposed to be either the receptor that recognizes and targets newly synthesized peroxisomal membrane proteins (PMP) to the peroxisome or a chaperone required for stabilization of PMPs at the peroxisomal membrane. Differentiating between these two roles for Pex19p could be achieved by determining whether the peroxisomal targeting signal (PTS) and the region of Pex19p binding of a PMP are the same or different. We addressed the role for Pex19p in the assembly of two PMPs, Pex30p and Pex32p, of the yeast Saccharomyces cerevisiae. Pex30p and Pex32p control peroxisome size and number but are dispensable for peroxisome formation. Systematic truncations from the carboxyl terminus, together with in-frame deletions of specific regions, have identified PTSs essential for targeting Pex30p and Pex32p to peroxisomes. Both Pex30p and Pex32p interact with Pex19p in regions that do not overlap with their PTSs. However, Pex19p is required for localizing Pex30p and Pex32p to peroxisomes, because mutations that disrupt the interaction of Pex19p with Pex30p and Pex32p lead to their mislocalization to a compartment other than peroxisomes. Mutants of Pex30p and Pex32p that localize to peroxisomes but produce cells exhibiting the peroxisomal phenotypes of cells lacking these proteins demonstrate that the regions in these proteins that control peroxisomal targeting and cell biological activity are separable. Together, our data show that the interaction of Pex19p with Pex30p and Pex32p is required for their roles in peroxisome biogenesis and are consistent with a chaperone role for Pex19p in stabilizing or maintaining membrane proteins in peroxisomes.
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ABSTRACT: During a search for genes controlling conidial dormancy in Aspergillus fumigatus, two dehydrin-like genes, DprA and DprB, were identified. The deduced proteins had repeated stretches of 23 amino acids that contained a conserved dehydrin-like protein (DPR) motif. Disrupted DprAΔ mutants were hypersensitive to oxidative stress and to phagocytic killing, whereas DprBΔ mutants were impaired in osmotic and pH stress responses. However, no effect was observed on their pathogenicity in our experimental models of invasive aspergillosis. Molecular dissection of the signaling pathways acting upstream showed that expression of DprA was dependent on the stress-activated kinase SakA and the cyclic AMP-protein kinase A (cAMP-PKA) pathways, which activate the bZIP transcription factor AtfA, while expression of DprB was dependent on the SakA mitogen-activated protein kinase (MAPK) pathway, and the zinc finger transcription factor PacC. Fluorescent protein fusions showed that both proteins were associated with peroxisomes and the cytosol. Accordingly, DprA and DprB were important for peroxisome function. Our findings reveal a novel family of stress-protective proteins in A. fumigatus and, potentially, in filamentous ascomycetes.Molecular biology of the cell 06/2011; 22(11):1896-906. DOI:10.1091/mbc.E10-11-0914 · 5.98 Impact Factor
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ABSTRACT: Biogenesis of the mammalian peroxisomal membrane requires the action of Pex3p and Pex16p, two proteins present in the organelle membrane, and Pex19p, a protein that displays a dual subcellular distribution (peroxisomal and cytosolic). Pex19p interacts with most peroxisomal intrinsic membrane proteins, but whether this property reflects its role as an import receptor for this class of proteins or a chaperone-like function in the assembly/disassembly of peroxisomal membrane proteins has been the subject of much controversy. Here, we describe an in vitro system particularly suited to address this issue. It is shown that insertion of a reporter protein into the peroxisomal membrane is a Pex3p-dependent process that does not require ATP/GTP hydrolysis. The system can be programmed with recombinant versions of Pex19p, allowing us to demonstrate that Pex19p-cargo protein complexes formed in the absence of peroxisomes are the substrates for the peroxisomal docking/insertion machinery. Data suggesting that cargo-loaded Pex19p displays a much higher affinity for Pex3p than Pex19p alone are also provided. These results suggest that soluble Pex19p participates in the targeting of newly synthesized peroxisomal membrane proteins to the organelle membrane and support the existence of a cargo-induced peroxisomal targeting mechanism for Pex19p.Journal of Biological Chemistry 12/2006; 281(45):34492-502. DOI:10.1074/jbc.M607183200 · 4.60 Impact Factor