New insights into import of proteins into peroxisomes
Although peroxisomes no longer appear to be autonomous organelles, they can nevertheless import their matrix proteins. A property that sets this machinery apart from most of the other import systems is the capacity to import (partially) folded proteins. Genetic screens have identified many of the proteins involved but functional insight into its workings has been hampered by a lack of a reconstituted in vitro system. Recently, Azevedo and co-workers have made important progress in this direction, allowing us to probe the initial steps of the process in action. A key observation was that 15% of the total Pex5p (the PTS1 receptor) is present in the peroxisomal fraction and behaves as an integral membrane protein that is associated with other known members of the protein import complex (Gouveia et al., 2000
; Reguenga et al., 2001). A protease-sensitivity assay has allowed Azevedo and co-workers to demonstrate conformational and/or topological changes in Pex5p related to its role in protein import. They have shown that entry of Pex5p into the membrane is cargo dependent but ATP independent, whereas return of Pex5p to the soluble phase does require ATP (Gouveia et al., 2003; Oliveira et al., 2003).
Azevedo and co-workers propose that cargo proteins are transported across the peroxisomal membrane by the PTS1 receptor itself (reviewed by Azevedo et al., 2004). To do this, Pex5p must behave both as a hydrophilic cytosolic protein and, temporarily, as a transmembrane protein. Erdmann and Schliebs have drawn an elegant analogy with pore-forming toxins (Erdmann and Schliebs, 2005). These also enter the membrane, self-associate and form a pore that mediates transport of various products, depending on the toxin involved. Erdmann and co-workers have reconstituted the Pex5p cycle in vitro, showing that return of Pex5p from the membrane into the soluble phase depends on Pex1p, Pex6p and Pex15p (Platta et al., 2005).
Pex15p (or Pex26 in mammalian cells) is an integral peroxisomal membrane protein that interacts with the soluble AAA ATPases Pex1p and Pex6p (Birschmann et al., 2003). Other proteins related to AAA ATPases include N-ethylmaleimide-sensitive fusion protein (NSF) and Cdc48/p97. NSF functions in N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-mediated membrane fusion, whereas Cdc48/p97 extracts proteins from the ER that are destined for ubiquitin-dependent degradation by the proteasome (Jarosch et al., 2002). This analogy between extraction of misfolded proteins from the ER and extraction of Pex5p from the peroxisomal membrane goes even further, because several groups have shown that Pex5p can be ubiquitylated (Platta et al., 2004; Kiel et al., 2005a; Kiel et al., 2005b; Kragt et al., 2005b). This might require Pex4p, a protein that resembles ubiquitin-conjugating enzymes. Pex4p binds to the membrane through Pex22p and the RING-finger membrane proteins Pex2p, Pex10p and Pex12p, which in turn resemble ubiquitin ligases (Platta et al., 2005). Both mono- and polyubiquitylation have been reported, and polyubiquitylated Pex5p is degraded by the proteasome. How and whether ubiquitylation is involved in the normal receptor-cycling process remains a matter for further investigation.
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