Suzanne Hoppins

A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria

Statement MITO-MAP, a high-density genetic interaction map in budding yeast, identifies a mitochondrial inner membrane–associated complex that promotes normal mitochondrial membrane organization and morphology.

Coassembly of Mgm1 isoforms requires cardiolipin and mediates mitochondrial inner membrane fusion

The soluble and membrane-anchored isoforms of Mgm1 are only active when they work together (in trans).

Tim50, a novel component of the TIM23 preprotein translocase of mitochondria

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. We isolated the TIM23 complex of Neurospora crassa. Besides Tim23 and Tim17, it contained a novel component, referred to as Tim50. Tim50 spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the intermembrane space. Tim50 is essential for viability of yeast. Mitochondria from cells depleted of Tim50 displayed strongly reduced import kinetics of preproteins using the TIM23 complex. Tim50 could be cross‐linked to preproteins that were halted at the level of the translocase of the outer membrane (TOM complex) or spanning both TOM and TIM23 complexes. We suggest that Tim50 plays a crucial role in the transfer of preproteins from the TOM complex to the TIM23 complex through the intermembrane space.

The Tim8-Tim13 complex of Neurospora crassa functions in the assembly of proteins into both mitochondrial membranes

The Tim8 and Tim13 proteins in yeast are known to exist in the mitochondrial intermembrane space and to form a hetero-oligomeric complex involved in the import of the mitochondrial inner membrane protein Tim23, the central component of the TIM23 translocase. Here, we have isolated tim8 and tim13 mutants in Neurospora crassa and have shown that mitochondria lacking the Tim8-Tim13 complex were deficient in the import of the outer membrane β-barrel proteins Tom40 and porin. Cross-linking studies showed that the Tom40 precursor contacts the Tim8-Tim13 complex. The complex is involved at an early point in the Tom40 assembly pathway because cross-links can only be detected during the initial stages of Tom40 import. In mitochondria lacking the Tim8-Tim13 complex, the Tom40 precursor appears in a previously characterized early intermediate of Tom40 assembly more slowly than in wild type mitochondria. Thus, our data suggest a model in which one of the first steps in Tom40 assembly may be interaction with the Tim8-Tim13 complex. As in yeast, the N. crassa Tim23 precursor was imported inefficiently into mitochondria lacking the Tim8-Tim13 complex when the membrane potential was reduced. Tim23 import intermediates could also be cross-linked to the complex, suggesting a dual role for the Tim8-Tim13 intermembrane space complex in the import of proteins found in both the outer and inner mitochondrial membranes.

Uniform nomenclature for the mitochondrial contact site and cristae organizing system

The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex “mitochondrial contact site and cristae organizing system” and its subunits Mic10 to Mic60.

The molecular mechanism of mitochondrial fusion.

This review is focused on mitochondrial membrane fusion, which is a highly conserved process from yeast to human cells. We present observations from both yeast and mammalian cells that have provided insights into the mechanism of mitochondrial fusion and speculate on how the key players, which are dynamin-related GTPases do the work of membrane tethering and fusion.

Mitochondrial Dynamics and Apoptosis—the ER Connection

Microdomains formed by the association of the ER with mitochondria during mitochondrial division may also be used to regulate cell death. Mitochondria are endosymbiotic organelles that were pivotal in the evolution of eukaryotic multicellular organisms, enabling cells, through production of adenosine 5′-triphosphate, to overcome a steep energetic barrier (1). Another essential feature of multicellularity is programmed cell death or apoptosis—a process in which mitochondria also play a critical role. During intrinsic apoptosis, a signaling platform assembles on mitochondria that in some organisms is harnessed to permeabilize the outer mitochondrial membrane and release proapoptotic proteins. Assembly of this platform is accompanied by dramatic changes in the dynamic behavior of mitochondria, which influence cell death. The dynamic properties of mitochondria are dependent on their division and fusion and govern the overall shape, connectedness, and distribution of mitochondria in cells. On page 1062 in this issue, Youle and van der Bliek (2) review the interplay between mitochondrial dynamics and mitochondrial quality-control and stress pathways. Here, we speculate on the role of mitochondrial division and fusion in the ultimate stress response, cell death. The recent discovery that the endoplasmic reticulum (ER), another ancient endomembrane organelle, actively participates in mitochondrial division has led to a new model linking mitochondrial dynamics and cell death. This suggests an unexpected convergence during evolution of mitochondria and ER—the two dominant endomembrane systems in eukaryotic cells that have previously been viewed as functionally distinct.

The regulation of mitochondrial dynamics

The structure of mitochondria is highly dynamic. Mitochondrial shape is cell-type specific and can be modified to meet changing requirements in energy production, calcium homeostasis, lipid biogenesis, fatty acid synthesis and other mitochondrial activities. This is achieved by modulating the dynamic properties of mitochondria including fusion, division, movement and positional tethering. It has become increasingly evident that mitochondrial dynamics also play an intimate role in several cellular signaling pathways and as such, many mechanisms have evolved to modulate mitochondrial structure. These regulatory mechanisms turn out to be important for modulation of mitochondrial-specific processes as well as cell, tissue and organism responses to developmental or environmental cues.

Mitochondrial outer and inner membrane fusion requires a modified carrier protein

In yeast, three proteins are essential for mitochondrial fusion. Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively. At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing. The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family. We show that Ugo1 is a modified member of this family, containing three transmembrane domains and existing as a dimer, a structure that is critical for the fusion function of Ugo1. Our functional analysis of Ugo1 indicates that it is required distinctly for both outer and inner membrane fusion after membrane tethering, indicating that it operates at the lipid-mixing step of fusion. This role is distinct from the fusion dynamin-related proteins and thus demonstrates that at each membrane, a single fusion protein is not sufficient to drive the lipid-mixing step, but instead, this step requires a more complex assembly of proteins.

Alternative Splicing Gives Rise to Different Isoforms of the Neurospora crassa Tob55 Protein That Vary in Their Ability to Insert β-Barrel Proteins Into the Outer Mitochondrial Membrane

Tob55 is the major component of the TOB complex, which is found in the outer membrane of mitochondria. A sheltered knockout of the tob55 gene was developed in Neurospora crassa. When grown under conditions that reduce the levels of the Tob55 protein, the strain exhibited a reduced growth rate and mitochondria isolated from these cells were deficient in their ability to import β-barrel proteins. Surprisingly, Western blots of wild-type mitochondrial proteins revealed two bands for Tob55 that differed by ∼4 kDa in their apparent molecular masses. Sequence analysis of cDNAs revealed that the tob55 mRNA is alternatively spliced and encodes three isoforms of the protein, which are predicted to contain 521, 516, or 483 amino acid residues. Mass spectrometry of proteins isolated from purified outer membrane vesicles confirmed the existence of each isoform in mitochondria. Strains that expressed each isoform of the protein individually were constructed. When cells expressing only the longest form of the protein were grown at elevated temperature, their growth rate was reduced and mitochondria isolated from these cells were deficient in their ability to assembly β-barrel proteins.