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.

Pex3p is a peroxisomal membrane protein that is essential for peroxisome biogenesis. Here, we show that a conserved stretch of positively charged amino acids (Arg 11 -X-Lys-Lys-Lys 15 ) in the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into its target membrane. Despite the strong conservation, this sequence shows a high degree of redundancy. Substitution of either Arg 11 , Lys 13 , Lys 14 , or Lys 15 with uncharged or negatively charged amino acids did not interfere with Pex3p location and function. However, a mutant Pex3p, carrying negatively charged amino acids at position 13 and 15 (K13E/K15E), caused moderate but significant defects in peroxisome assembly and matrix protein import. Additional changes in the N terminus of Pex3p, e.g. replacing three or four of the positively charged amino acids with negatively charged ones, led to a typical pex3 phenotype, i.e. accumulation of peroxisomal matrix proteins in the cytosol and absence of peroxisomal remnants. Also, in these cases, the mutant Pex3p levels were reduced. Remarkably, mutant Pex3p proteins were mislocalized to mitochondria or the cytosol, depending on the nature of the mutation. Furthermore, in case of reduced amounts of Pex3p, the levels of other peroxisomal membrane proteins, e.g. Pex10p and Pex14p, were also diminished, suggesting that Pex3p maybe involved in the recruitment or stabilization of these proteins (in the membrane).

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.

Contrary to earlier findings, preperoxisomal membrane structures form in yeast cells lacking the peroxin Pex3 and are competent to mature into functional peroxisomes upon Pex3 reintroduction.

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 Pex11β 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.