Controlling peroxisome-ER contacts – Molecular mechanisms to regulate peroxisome tethering and distribution

Abstract

Peroxisomes are small, single membrane bound organelles with key roles in cellular lipid and hydrogen peroxide metabolism. They contribute to a wide range of metabolic processes including the β-oxidation of fatty acids and the synthesis of ether-phospholipids. To fulfil those functions, peroxisomes cooperate with other organelles, such as the endoplasmic reticulum (ER) and mitochondria. This collaboration requires close proximity of the organelles, which is mediated by protein tethering complexes that physically bridge apposing organelles. This thesis focuses on the interaction of peroxisomes with the ER that are mediated through interaction of the peroxisomal membrane protein ACBD5 and the ER-resident protein VAPB. These peroxisome-ER contacts are important for peroxisome motility, and the transfer of membrane lipids and ether-phospholipid intermediates. ACBD5 binds to VAPB via its FFAT motif. However, molecular mechanisms, which regulate formation of these contact sites, are unknown. Additionally, ACBD5 deficient patients present with neurological problems, but the pathological mechanisms are not well understood. Here, I explore the regulation of peroxisome-ER contacts and study them in model organisms. My findings reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. I show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT motif, which alter interaction with VAPB – and thus peroxisome-ER contact sites – differently. Moreover, I demonstrate that the kinase GSK3β regulates this interaction. These results reveal for the first time a molecular mechanism for the regulation of peroxisome-ER contacts in mammalian cells and expand the current model of the FFAT-VAP interaction. In addition, I used two model systems, the fruit fly Drosophila melanogaster and the filamentous fungus Ustilago maydis, to study the impact of ACBD5 depletion on peroxisome dynamics in vivo in long polarized neurites and hyphae, respectively. I discovered that the D. melanogaster ACBD5 and VAP homologs interact, while the U. maydis proteins do not, but depletion of ACBD5 in both species leads to redistribution of peroxisomes. Overall, this provides valuable findings to increase our understanding of the pathophysiologic processes in ACBD5 deficient patients.