Catherine S. Palmer

MiD49 and MiD51, new components of the mitochondrial fission machinery

Mitochondria form intricate networks through fission and fusion events. Here, we identify mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) anchored in the mitochondrial outer membrane. MiD49/51 form foci and rings around mitochondria similar to the fission mediator dynamin‐related protein 1 (Drp1). MiD49/51 directly recruit Drp1 to the mitochondrial surface, whereas their knockdown reduces Drp1 association, leading to unopposed fusion. Overexpression of MiD49/51 seems to sequester Drp1 from functioning at mitochondria and cause fused tubules to associate with actin. Thus, MiD49/51 are new mediators of mitochondrial division affecting Drp1 action at mitochondria.

The regulation of mitochondrial morphology: intricate mechanisms and dynamic machinery.

Mitochondria typically form a reticular network radiating from the nucleus, creating an interconnected system that supplies the cell with essential energy and metabolites. These mitochondrial networks are regulated through the complex coordination of fission, fusion and distribution events. While a number of key mitochondrial morphology proteins have been identified, the precise mechanisms which govern their activity remain elusive. Moreover, post translational modifications including ubiquitination, phosphorylation and sumoylation of the core machinery are thought to regulate both fusion and division of the network. These proteins can undergo several different modifications depending on cellular signals, environment and energetic demands of the cell. Proteins involved in mitochondrial morphology may also have dual roles in both dynamics and apoptosis, with regulation of these proteins under tight control of the cell to ensure correct function. The absolute reliance of the cell on a functional mitochondrial network is highlighted in neurons, which are particularly vulnerable to any changes in organelle dynamics due to their unique biochemical requirements. Recent evidence suggests that defects in the shape or distribution of mitochondria correlate with the progression of neurodegenerative diseases such as Alzheimers, Huntingtons and Parkinsons disease. This review focuses on our current understanding of the mitochondrial morphology machinery in cell homeostasis, apoptosis and neurodegeneration, and the post translational modifications that regulate these processes.

Recent advances into the understanding of mitochondrial fission

Mitochondria exist as a highly dynamic tubular network, and their morphology is governed by the delicate balance between frequent fusion and fission events, as well as by interactions with the cytoskeleton. Alterations in mitochondrial morphology are associated with changes in metabolism, cell development and cell death, whilst several human pathologies have been associated with perturbations in the cellular machinery that coordinate these processes. Mitochondrial fission also contributes to ensuring the proper distribution of mitochondria in response to the energetic requirements of the cell. The master mediator of fission is Dynamin related protein 1 (Drp1), which polymerises and constricts mitochondria to facilitate organelle division. The activity of Drp1 at the mitochondrial outer membrane is regulated through post-translational modifications and interactions with mitochondrial receptor and accessory proteins. This review will concentrate on recent advances made in delineating the mechanism of mitochondrial fission, and will highlight the importance of mitochondrial fission in health and disease. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.

Adaptor proteins MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and are specific for mitochondrial fission.

Background: Various receptor proteins recruit Drp1 to drive fission of mitochondria and peroxisomes. Results: MiD49 and MiD51 recruit Drp1 specifically to mitochondria independently of receptors Fis1 and Mff. Conclusion: MiD49 and MiD51 appear to be specific to the mitochondrial fission apparatus of mammalian cells. Significance: Mitochondrial and peroxisomal fission processes can be differentially regulated. Drp1 (dynamin-related protein 1) is recruited to both mitochondrial and peroxisomal membranes to execute fission. Fis1 and Mff are Drp1 receptor/effector proteins of mitochondria and peroxisomes. Recently, MiD49 and MiD51 were also shown to recruit Drp1 to the mitochondrial surface; however, different reports have ascribed opposing roles in fission and fusion. Here, we show that MiD49 or MiD51 overexpression blocked fission by acting in a dominant-negative manner by sequestering Drp1 specifically at mitochondria, causing unopposed fusion events at mitochondria along with elongation of peroxisomes. Mitochondrial elongation caused by MiD49/51 overexpression required the action of fusion mediators mitofusins 1 and 2. Furthermore, at low level overexpression when MiD49 and MiD51 form discrete foci at mitochondria, mitochondrial fission events still occurred. Unlike Fis1 and Mff, MiD49 and MiD51 were not targeted to the peroxisomal surface, suggesting that they specifically act to facilitate Drp1-directed fission at mitochondria. Moreover, when MiD49 or MiD51 was targeted to the surface of peroxisomes or lysosomes, Drp1 was specifically recruited to these organelles. Moreover, the Drp1 recruitment activity of MiD49/51 appeared stronger than that of Mff or Fis1. We conclude that MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and suggest that they provide specificity to the division of mitochondria.

Cooperative and independent roles of the Drp1 adaptors Mff, MiD49 and MiD51 in mitochondrial fission

ABSTRACT Cytosolic dynamin-related protein 1 (Drp1, also known as DNM1L) is required for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used gene-editing technology to create a suite of cell lines lacking the adaptors MiD49 (also known as MIEF2), MiD51 (also known as MIEF1), Mff and Fis1. Increased mitochondrial connectivity was observed following loss of individual adaptors, and this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49 and MiD51 showing the more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we found that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission. Highlighted Article: MiD51 and Mff coordinate and regulate the action of Drp1 at the mitochondrial outer membrane during homeostatic mitochondrial fission, as assessed with gene-editing technology.

Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission.

Structure–function analyses driven by a crystal structure of the cytosolic domain of the Drp1 receptor MiD51 reveals a nucleotidyltransferase fold and nucleotide binding activity that is independent of its Drp1 binding activity.

Structural and Functional Requirements for Activity of the Tim9–Tim10 Complex in Mitochondrial Protein Import

The Tim9-Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9-Tim10 hexameric assembly determined to 2.5 A and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.

Mitochondrial protein quality control in health and disease

Progressive mitochondrial dysfunction is linked with the onset of many age‐related pathologies and neurological disorders. Mitochondrial damage can come in many forms and be induced by a variety of cellular insults. To preserve organelle function during biogenesis or times of stress, multiple surveillance systems work to ensure the persistence of a functional mitochondrial network. This review provides an overview of these processes, which collectively contribute to the maintenance of a healthy mitochondrial population, which is critical for cell physiology and survival.

Spatial organization of protein export in malaria parasite blood stages

Plasmodium falciparum, which causes malaria, extensively remodels its human host cells, particularly erythrocytes. Remodelling is essential for parasite survival by helping to avoid host immunity and assisting in the uptake of plasma nutrients to fuel rapid growth. Host cell renovation is carried out by hundreds of parasite effector proteins that are exported into the erythrocyte across an enveloping parasitophorous vacuole membrane (PVM). The Plasmodium translocon for exported (PTEX) proteins is thought to span the PVM and provide a channel that unfolds and extrudes proteins across the PVM into the erythrocyte. We show that exported reporter proteins containing mouse dihydrofolate reductase domains that inducibly resist unfolding become trapped at the parasite surface partly colocalizing with PTEX. When cargo is trapped, loop‐like extensions appear at the PVM containing both trapped cargo and PTEX protein EXP2, but not additional components HSP101 and PTEX150. Following removal of the block‐inducing compound, export of reporter proteins only partly recovers possibly because much of the trapped cargo is spatially segregated in the loop regions away from PTEX. This suggests that parasites have the means to isolate unfoldable cargo proteins from PTEX‐containing export zones to avert disruption of protein export that would reduce parasite growth.

The Rare Anaphylaxis-Associated FcγRIIa3 Exhibits Distinct Characteristics From the Canonical FcγRIIa1

FcγRIIa is an activating FcγR, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as FcγRIIa1. A unique splice variant, we designated FcγRIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this FcγRIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. FcγRIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. FcγRIIa3 was identified in macaque and human PBMC. The FcγRIIa3 is distinguished from the canonical FcγRIIa1 by a unique 19-amino acid cytoplasmic insertion and these two FcγRIIa forms responded distinctly to antibody ligation. Whereas FcγRIIa1 was rapidly internalized, FcγRIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four FCGR2A SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of FcγRIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced FcγRIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.