Peroxisomal membrane protein degradation in yeast

Peroxisomes are single membrane-bound organelles that are present in nearly all eukaryotic cells. Some well-known peroxisome functions include β-oxidation of fatty acids, synthesis of plasmalogens and detoxification of hydrogen peroxide, but many more exist. Peroxisomal functions are largely determined by peroxisomal matrix proteins and peroxisomal membrane proteins (PMPs). Thus, peroxisomal proteins need to be tightly controlled through regulation of protein synthesis, targeting as well as protein degradation. Peroxisomal proteins may undergo degradation either because they are damaged/unfolded by various stress factors or because they are no longer needed. Protein degradation needs to be tightly controlled in order to prevent the accumulation of unwanted proteins or the occurrence of unwanted protein degradation events. While many efforts have been placed on studying the post-translational targeting of PMPs to peroxisomes, little is known about PMP degradation. Investigating which PMPs are targeted for degradation, how and why PMPs are degraded will allow us to better understand the role of PMP degradation in a cellular context. The research presented in this thesis provides insights into PMP degradation in the yeast Saccharomyces cerevisiae. Using the tandem fluorescent timer (tFT) approach which employs two fluorescent proteins of different maturation kinetics, we investigated the stability of PMP-tFT fusions and identified two unstable proteins (Pxa1-tFT and Pex13-tFT) that are rapidly degraded from peroxisomes. Furthermore using the same approach, we also identified for the first time that in addition to peroxisomal Ubiquitin Proteasome System (UPS) components, cytosolic proteins also function in the degradation of unstable PMP-tFT fusions as well as a mutant form of Pxa1p. Lastly, we show that like other organelles such as ER, peroxisomes possess a quality control system to remove faulty proteins

Insights into the Role of the Peroxisomal Ubiquitination Machinery in Pex13p Degradation in the Yeast Hansenula polymorpha

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