Role of Cdc48p and Msp1p in Peroxisomal Protein Quality Control and Communication

• Applicant: Professor Dr. Ralf Erdmann
• Subject Area: Biochemistry
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 528852166

Peroxisomes are single membrane-bound cell organelles that are very special as 1) they import folded, even oligomeric proteins and 2) this import is performed by receptors that cycle between the cytosol and the peroxisomal membrane. Most peroxisomal matrix enzymes are imported by the import receptor Pex5p. Central steps in the receptor cycle of Pex5p comprise the recognition of newly synthesized proteins in the cytosol followed by receptor/cargo assembly with the importomer at the peroxisomal membrane. Following cargo release, Pex5p is monoubiquitinated and released from the membrane by the AAA-type ATPases Pex1p and Pex6p in an ATP-dependent manner. A defect in receptor recycling would result in a block of the import machinery. This is avoided by a quality-control system that clears the peroxisome membrane of accumulating receptors. This system, termed RADAR (Receptor Accumulation and Degradation in Absence of Recycling), is conserved among species, and leads to the extraction of the receptor from the membrane and its proteasomal degradation. However, our knowledge on the RADAR pathway is scarce. In this project, we aim to identify the factors that are required for the RADAR pathway as well as the quality control of other peroxins by using baker’s yeast as a model organism. To this end, we will pursue the following main goals: 1) Based on our preliminary data demonstrating that the AAA-ATPase Cdc48p plays a major role in this process, the first goal of this project is to elucidate the molecular function of Cdc48p for peroxisome function and maintenance. We will investigate whether known Cdc48p adaptor proteins are required for RADAR. We will reconstitute the export process in vitro and identify crucial amino acid residues with focus on the role of ubiquitination. Finally, we will isolate and characterize the peroxisomal and cytosolic RADAR-associated machineries. 2) The second goal of this project addresses the AAA-ATPase Msp1p, which is known for its role in the quality control of mitochondrial and peroxisomal tail-anchored membrane proteins. Our initial data indicate that the substrate spectrum of Msp1p is not limited to tail-anchored proteins but also comprises other kind of integral membrane proteins, including proteins with multiple membrane spans. Here we intend to apply systematic approaches to identify novel Msp1p-targets and thus to investigate whether Msp1 performs a more general role in the quality control of peroxisomal membrane proteins. 3) Based on our observation that peroxisomes and mitochondria cluster upon overexpression of non-functional Msp1p, we intend to isolate and characterize the corresponding organelle tethers.

• DFG Programme: Research Grants