The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission.

The initial phase of peroxisomal fission requires the peroxisomal membrane protein Peroxin 11 (Pex11p), which remodels the membrane, resulting in organelle elongation. Here, we identify an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: dynamin-like protein (DLP)-mediated membrane scission. First, we demonstrate that yeast Pex11p is necessary for the function of the GTPase Dynamin-related 1 (Dnm1p) in vivo. In addition, our data indicate that Pex11p physically interacts with Dnm1p and that inhibiting this interaction compromises peroxisomal fission. Finally, we demonstrate that Pex11p functions as a GTPase activating protein (GAP) for Dnm1p in vitro. Similar observations were made for mammalian Pex11ß and the corresponding DLP Drp1, indicating that DLP activation by Pex11p is conserved. Our work identifies a previously unknown requirement for a GAP in DLP function.

Yeast pex1 cells contain peroxisomal ghosts that import matrix proteins upon reintroduction of Pex1.

Pex1 and Pex6 are two AAA-ATPases that play a crucial role in peroxisome biogenesis. We have characterized the ultrastructure of the Saccharomyces cerevisiae peroxisome-deficient mutants pex1 and pex6 by various high-resolution electron microscopy techniques. We observed that the cells contained peroxisomal membrane remnants, which in ultrathin cross sections generally appeared as double membrane rings. Electron tomography revealed that these structures consisted of one continuous membrane, representing an empty, flattened vesicle, which folds into a cup shape. Immunocytochemistry revealed that these structures lack peroxisomal matrix proteins but are the sole sites of the major peroxisomal membrane proteins Pex2, Pex10, Pex11, Pex13, and Pex14. Upon reintroduction of Pex1 in Pex1-deficient cells, these peroxisomal membrane remnants (ghosts) rapidly incorporated peroxisomal matrix proteins and developed into peroxisomes. Our data support earlier views that Pex1 and Pex6 play a role in peroxisomal matrix protein import.

Central role of Mic10 in the mitochondrial contact site and cristae organizing system.

The mitochondrial contact site and cristae organizing system (MICOS) is a conserved multi-subunit complex crucial for maintaining the characteristic architecture of mitochondria. Studies with deletion mutants identified Mic10 and Mic60 as core subunits of MICOS. Mic60 has been studied in detail; however, topogenesis and function of Mic10 are unknown. We report that targeting of Mic10 to the mitochondrial inner membrane requires a positively charged internal loop, but no cleavable presequence. Both transmembrane segments of Mic10 carry a characteristic four-glycine motif, which has been found in the ring-forming rotor subunit of F1Fo-ATP synthases. Overexpression of Mic10 profoundly alters the architecture of the inner membrane independently of other MICOS components. The four-glycine motifs are dispensable for interaction of Mic10 with other MICOS subunits but are crucial for the formation of large Mic10 oligomers. Our studies identify a unique role of Mic10 oligomers in promoting the formation of inner membrane crista junctions.

Preperoxisomal vesicles can form in the absence of Pex3.

We demonstrate that the peroxin Pex3 is not required for the formation of peroxisomal membrane structures in yeast pex3 mutant cells. Notably, pex3 mutant cells already contain reticular and vesicular structures that harbor key proteins of the peroxisomal receptor docking complex-Pex13 and Pex14-as well as the matrix proteins Pex8 and alcohol oxidase. Other peroxisomal membrane proteins in these cells are unstable and transiently localized to the cytosol (Pex10, Pmp47) or endoplasmic reticulum (Pex11). These reticular and vesicular structures are more abundant in cells of a pex3 atg1 double deletion strain, as the absence of Pex3 may render them susceptible to autophagic degradation, which is blocked in this double mutant. Contrary to earlier suggestions, peroxisomes are not formed de novo from the endoplasmic reticulum when the PEX3 gene is reintroduced in pex3 cells. Instead, we find that reintroduced Pex3 sorts to the preperoxisomal structures in pex3 cells, after which these structures mature into normal peroxisomes.

Role of MINOS in mitochondrial membrane architecture: cristae morphology and outer membrane interactions differentially depend on mitofilin domains.

The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein complexes of the mitochondrial outer membrane. To study if outer membrane interactions and maintenance of cristae morphology are directly coupled, we generated mutant forms of mitofilin/Fcj1 (formation of crista junction protein 1), a core component of MINOS. Mitofilin consists of a transmembrane anchor in the inner membrane and intermembrane space domains, including a coiled-coil domain and a conserved C-terminal domain. Deletion of the C-terminal domain disrupted the MINOS complex and led to release of cristae membranes from the inner boundary membrane, whereas the interaction of mitofilin with the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM) were enhanced. Deletion of the coiled-coil domain also disturbed the MINOS complex and cristae morphology; however, the interactions of mitofilin with TOM and SAM were differentially affected. Finally, deletion of both intermembrane space domains disturbed MINOS integrity as well as interactions with TOM and SAM. Thus, the intermembrane space domains of mitofilin play distinct roles in interactions with outer membrane complexes and maintenance of MINOS and cristae morphology, demonstrating that MINOS contacts to TOM and SAM are not sufficient for the maintenance of inner membrane architecture.

Normal peroxisome development from vesicles induced by truncated Hansenula polymorpha Pex3p.

We show that the synthesis of the N-terminal 50 amino acids of Pex3p (Pex3p(1-50)) in Hansenula polymorpha pex3 cells is associated with the formation of vesicular membrane structures. Biochemical and ultrastructural findings suggest that the nuclear membrane is the donor membrane compartment of these vesicles. These structures also contain Pex14p and can develop into functional peroxisomes after subsequent reintroduction of the full-length Pex3p protein. We discuss the significance of this finding in relation to peroxisome reintroduction, e.g. in case peroxisomes are lost due to failure in inheritance.

Isolation of mutants of Hansenula polymorpha defective in the assembly of octameric alcohol oxidase.

Alcohol oxidase (AO) is a peroxisomal enzyme that catalyses the first step in methanol metabolism in yeast. Monomeric, inactive AO protein is synthesised in the cytosol and subsequently imported into peroxisomes, where the enzymatically active, homo-octameric form is found. The mechanisms involved in AO octamer assembly are largely unclear. Here we describe the isolation of Hansenula polymorpha mutants specifically affected in AO assembly. These mutants are unable to grow on methanol and display reduced AO activities. Based on their phenotypes, three major classes of mutants were isolated. Three additional mutants were isolated that each displayed a unique phenotype. Complementation analysis revealed that the isolated AO assembly mutants belonged to 10 complementation groups.