Anita M. Kram

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.

Brefeldin A interferes with peroxisomal protein sorting in the yeast Hansenula polymorpha

We have studied the effect of brefeldin A (BFA), a fungal toxin that interferes with coated vesicle formation, on the biogenesis of peroxisomes in the yeast Hansenula polymorpha. Addition of BFA (20 μg/ml) to cultures of H. polymorpha partially inhibited the development of peroxisomes and resulted in the reversible accumulation of newly synthesized peroxisomal membrane and matrix proteins at the endoplasmic reticulum. In contrast, BFA did not interfere with the selective degradation of peroxisomes. Taken together, our data suggest that the ER plays a crucial role in peroxisome biogenesis in H. polymorpha, possibly in the biosynthesis of the peroxisomal membrane.

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 (Pex3p1–50) 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.

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

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

Significance Peroxisomal fission is crucial for cell viability because peroxisome fission defects cause severe disease. The initial step in peroxisomal fission, membrane elongation, requires the membrane remodeling protein Peroxin 11 (Pex11p). Here, we identify an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: membrane separation. We show that Pex11p functions as a GTPase activating protein (GAP) for Dynamin-related 1 (Dnm1p) and that this GAP activity is conserved from yeast to mammalians. This work identifies a previously unknown requirement for a GAP in dynamin-like protein function. 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.

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

Yeast Pex1- and Pex6-deficient cells harbor peroxisomal membrane ghosts that lack matrix proteins but contain all major peroxisomal membrane proteins and acquire matrix proteins to develop into normal peroxisomes upon Pex1 reintroduction.