Peroxisome biogenesis initiates at the endoplasmic reticulum (ER) and maturation allows for the formation of metabolically active organelles. Yet, peroxisomes can also multiply by growth and division. Several proteins, called peroxins, are known to participate in these processes but little is known about their organization to orchestrate peroxisome proliferation. Here, we demonstrate that regulation of peroxisome proliferation relies on the integrity of the tubular ER network. Using a dual track SILAC-based quantitative interaction proteomics approach, we established a comprehensive network of stable as well as transient interactions of the peroxin Pex30p, an integral membrane protein. Through association with merely ER resident proteins, in particular with proteins containing a reticulon homology domain, and with other peroxins, Pex30p designates peroxisome contact sites at ER subdomains. We show that Pex30p traffics through the ER and segregates in punctae to which peroxisomes specifically append, and we ascertain its transient interaction with all subunits of the COPI coatomer complex suggesting the involvement of a vesicle-mediated transport. We establish that the membrane protein Pex30p facilitates the connection of peroxisomes to the ER. Taken together, our data indicate that Pex30p-containing protein complexes act as focal points from which peroxisomes can form and that the tubular ER architecture organized by the reticulon homology proteins Rtn1p, Rtn2p and Yop1p controls this process.All nucleated cells contain essential round-shaped organelles called peroxisomes, whose function is mainly associated with lipid metabolism (
1). Depending on the cellular requirements, the size, number, and protein content of these single membrane-bound organelles can vary widely. Although peroxisomes are dispensable for unicellular species such as yeasts, they are essential for the development of multicellular organisms (
2,
3). In human, mutations in
PEX genes lead to defects in peroxisome function or formation and are associated with the development of lethal pathologies (
4). These
PEX genes code for proteins, called peroxins, which are involved in peroxisome assembly and maintenance (
5).Two major routes seem to lead to peroxisome formation, namely,
de novo biogenesis and growth/division of pre-existing peroxisomes. The division pathway operates with proteins of the Pex11 family and requires fission factors shared with mitochondria (
6). Studies in yeast and mammalian cells revealed that through the action of the protein Pex3p peroxisome precursors can also originate from the endoplasmic reticulum (ER)
1 and, via import of membrane and matrix proteins, mature into fully functional organelles (
7,
8). Furthermore, several peroxisomal membrane proteins were shown to migrate to peroxisomes via the ER (
7,
9,
10). The molecular mechanism underlying the biogenic pathway of peroxisome formation has not been clarified so far. Recent data based on cell-free vesicle-budding reactions, however, demonstrated that several peroxisomal proteins traffic from the ER to peroxisomes in a COPII vesicle-independent manner (
11). These observations point to the existence of vesicular events to mediate the transport of peroxisomal membrane proteins from the ER. In fact, analysis of secretory mutant yeast cells already suggest that part of the ER-associated secretory machinery is involved in peroxisome biogenesis (
12).The
de novo biogenesis of peroxisomes and the growth/division pathways are usually seen as independent routes; however, these events may be coordinated and, thus, intimately linked. Indeed, peroxisomes need to acquire membrane components to proliferate and it has been proposed that their binding to the cell cortex or to the cytoskeleton allows their partitioning and segregation during cell division (
13–
15).Among the proteins required for assembly of peroxisomes, the membrane proteins Pex23p and Pex24p play essential roles in the yeast
Yarrowia lipolytica (
16,
17). Homologs of these two proteins in
Saccharomyces cerevisiae are Pex30p, Pex31p, and Pex32p, all containing at least one transmembrane domain and a dysferlin domain as common structural motifs, as well as Pex28p and Pex29p. In
S. cerevisiae, these proteins seem to negatively control peroxisomal size and number (
18,
19). Interestingly, Pex30p seems to exhibit species-specific differences in the regulation of peroxisome proliferation. While the lack of Pex30p in
S. cerevisiae leads to an increase in the number of normal-sized peroxisomes (
18), in
Pichia pastoris its absence correlates with the appearance of fewer and clustered peroxisomes (
20). Although peroxisomes are highly versatile organelles, under given conditions their total number per cell remains fairly constant owing to the delicate balance of proliferation, inheritance and degradation (
21,
22). The question is: what are the molecular mechanisms responsible for the spatiotemporal organization of these events?Here, we present data obtained from a dual approach based on quantitative interaction proteomics using stable isotope labeling with amino acids in cell culture (SILAC) (
23,
24) and live-cell imaging, revealing for the first time the dynamic interaction network around Pex30p and its function in the organization of ER-to-peroxisome membrane associations. We report the existence of a macromolecular membrane protein complex that acts as a hub for the regulation of peroxisome proliferation and movement. Our data suggest a direct role for the tubular cortical ER and the reticulon homology proteins Rtn1p, Rtn2p, and Yop1p in the regulation of peroxisome biogenesis. Furthermore, as an initially cortical-ER localized protein that interacts with reticulon homology proteins, Pex30p is shown in this work to establish contacts between ER tubules and peroxisomes and to specifically traffic through the ER. In summary, our data reveal a central role for Pex30p in the formation of ER-to-peroxisomes associations that appear to be involved in the coordination of peroxisome biogenesis and maintenance.
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