The import of matrix proteins into peroxisomes in yeast requires the action of the ubiquitin-conjugating enzyme Pex4p and a complex consisting of the ubiquitin E3 ligases Pex2p, Pex10p and Pex12p. Together, this peroxisomal ubiquitination machinery is thought to ubiquitinate the cycling receptor protein Pex5p and members of the Pex20p family of co-receptors, a modification that is required for receptor recycling. However, recent reports have demonstrated that this machinery plays a role in additional peroxisome-associated processes. Hence, our understanding of the function of these proteins in peroxisome biology is still incomplete. Here, we identify a role for the peroxisomal ubiquitination machinery in the degradation of the peroxisomal membrane protein Pex13p. Our data demonstrate that Pex13p levels build up in cells lacking members of this machinery and also establish that Pex13p undergoes rapid degradation in wild-type cells. Furthermore, we show that Pex13p is ubiquitinated in wild-type cells and also establish that Pex13p ubiquitination is reduced in cells lacking a functional peroxisomal E3 ligase complex. Finally, deletion of PEX2 causes Pex13p to build up at the peroxisomal membrane. Taken together, our data provide further evidence that the role of the peroxisomal ubiquitination machinery in peroxisome biology goes much deeper than receptor recycling alone.

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The knowns and unknowns of peroxisomal membrane protein degradation Chapter 1 18 The knowns and unknowns of Peroxisomal membrane protein degradation 21 1 Often, E2 enzymes directly transfer Ub to a target protein, typically with the help of E3s such as really interesting new gene (RING) domain containing ligases, also called as RING E3s (Figure 1, point 1) (Ozkan et al., 2005). However in some cases, the Ub is first transferred from the E2 to the active site of a specific class of E3 (Figure 1, point 2), which possess homologous to the E6-AP carboxyl terminus (HECT) domain and these HECT E3s are responsible for attaching Ub to the lysine residue of the target protein (Metzger et al., 2012; Petroski et al., 2006). A third class of E3 ligase, distinct from the RING and HECT classes, are referred to as RING-in-between-RING (RBR) E3s (Figure 1, point 3) and they often contain a RING domain, followed by an RBR domain and finally a RING-like domain. These RBR E3s recruit an E2, which transfers ubiquitin to a cysteine in the RING-like domain before transferring to the substrate (Figure 1, point 3) (Wenzel et al., 2011). Figure 1: Schematic overview of the enzyme cascade catalyzing protein ubiquitination and degradation. Ubiquitin (Ub) is first activated by a ubiquitin activating enzyme (E1) with ATP hydrolysis. Next, the E1 transfers the Ub to a ubiquitin conjugating enzyme (E2), which is eventually transferred to a substrate with the aid of a ubiquitin ligase (E3). (1) RING-E3 serve as a bridge to enable Ub to be passed directly from the E2 to the substrate while (2) HECT-E3s accept Ub from the E2 onto their own active site before attaching it to a substrate. Chapter 1 22 RBR-E3 ligases (3) bind a Ub conjugated E2 and Ub is transferred from the E2 to the E3, after which Ub is then transferred to the substrate. Substrate proteins with single Ub can undergo several rounds of Ub chain elongation. Poly-ubiquitinated substrates are then targeted to the proteasome for degradation. Cells possess one or two E1s, several E2s (~11 in yeast and ~37 in humans) and multiple E3s (~50 E3s in yeast and ~1000/thousand in humans) (