Morphogenic proteins and organelle homeostasis

Understanding the biological function of membrane proteins requires the knowledge of their in vivo topology and interactions. The research goals of the proposed project aim to better understand biological systems at the level of the dynamic process of organellar endomembrane proliferation. Cellular compartmentalization requires membrane- surrounded structures. Maintenance of the cellular endomembrane system homeostasis has to be tightly regulated according to the metabolic status of the cell and is therefore, essential for eukaryotic life. Hence, organelles are amenable to extra- and intracellular variations to harmonize the cellular metabolism and participate to the establishment of the tolerance to various detrimental assaults. Defect in the function of sub-cellular organelles is critically involved in the development of devastating human diseases. Consequently, we aim to scrutinize the macromolecular architecture of a protein system involved in regulating endomembrane proliferation. Peroxisome function mostly comprises lipid metabolism and these organelles have emerged as significant contributors to cellular oxidative stress through their ability to generate and degrade hydrogen peroxide and other reactive oxygen species, which may connect their function to the process of aging. Still, information on the mechanism by which they proliferation is incomplete. While 32 proteins, the peroxins, have been identified that are involved in peroxisome biogenesis only a few have been linked to the process of peroxisomal endomembrane proliferation. Among the latter, members of the Pex23-family are regarded as negative regulators of peroxisome size and number. We have previously found that Pex30 a membrane protein member of the Pex23-family associates with Yop1 and reticulon proteins. These factors have recently been described as morphogenic proteins necessary to form and maintain the tubular endoplamic reticulum (ER) suggesting a link between peroxisome endomembrane proliferation and ER tubules. Here we will establish the connection between components of the ER and proliferation of the peroxisomal endomembrane system by analyzing the spatiotemporal dynamics of morphogenic proteins in yeast and human cells. To this endeavor, we will examine interaction partners, pinpoint functional motifs and identify components of common complexes via mass spectrometry. To understand the exact function morphogenic proteins in peroxisome proliferation we will follow their dynamics using biochemical as well as live-cell imaging techniques upon optimal growth conditions versus oxidative stress. The results of our study will lead to a better understanding of the dynamics of the cellular endomembrane system and its vulnerability to oxidative insults. This will serve to comprehend the function of morphogenic proteins in the process of organellar endomenbrane proliferation and may provide new grounds for hypothesis about how organelles are formed and function.

Nucleated cells are divided into specialized compartments called the organelles. Among all organelles, peroxisomes are especially important for the metabolism of fat. In human, mutations in PEX genes lead to defects in peroxisomal function or formation. PEX genes code for proteins, the peroxins, which are necessary for the assembly and formation of peroxisomes and their mutations are associated with severe neurological disorders. Peroxisomes are formed (i) by duplication through growth/division with proteins belonging to the PEX11 family and fission factors shared with mitochondria and, (ii) through budding from the endoplasmic reticulum (ER). In this project, we analyzed the molecular mechanisms underlying peroxisome maintenance in yeasts and human cells. We employed a dual approach based on quantitative proteomics and live-imaging to analyze molecular networks involved in the regulation of peroxisome formation. The results of our studies in the yeast Saccharomyces cerevisiae point to a direct role for the peroxisome proliferation factor Pex30p in the establishment of ER-to- peroxisome associations. Furthermore, our data suggest that ER resident proteins belonging to the reticulon family, Rtn1p, Rtn2p and Yop1p may participate in the regulation of peroxisome formation. Pex30p seems to coordinate peroxisome proliferation as mediator of anchorage of peroxisome to the ER membrane. We also characterized a human ortholog of the yeast Pex30p. Using a functional assay to study the molecular details of peroxisome proliferation in living cells, we demonstrated that mammalian PEX11 proteins act as membrane elongation factors. Analysis of mutated PEX11 proteins revealed that during peroxisome duplication, while the matrix content of the template organelle remains sequestered, the new peroxisome acquires novel proteins. This mechanism may lead to rejuvenation of the peroxisome pool in the cell. Our studies demonstrated the function of PEX11 proteins in peroxisome polarization and membrane protrusion. Division of organelles requires membrane remodeling events to enable and facilitate the assembly of the fission machinery. Our data provide evidence that elongation of the peroxisomal membrane through the action of PEX11 proteins escorts the recruitment of mitochondrial fission factors to the peroxisomal membrane and membrane constriction allowing for division of the organelle. Our studies in yeast, plant and human cells established the evolutionary conservation of peroxisome proliferation machinery.