Domain Interactions within Fzo1 Oligomers Are Essential for Mitochondrial Fusion

Abstract

Mitofusins are conserved GTPases essential for the fusion of mitochondria. These mitochondrial outer membrane proteins contain a GTPase domain and two or three regions with hydrophobic heptad repeats, but little is known about how these domains interact to mediate mitochondrial fusion. To address this issue, we have analyzed the yeast mitofusin Fzo1p and find that mutation of any of the three heptad repeat regions (HRN, HR1, and HR2) leads to a null allele. Specific pairs of null alleles show robust complementation, indicating that functional domains need not exist on the same molecule. Biochemical analysis indicates that this complementation is due to Fzo1p oligomerization mediated by multiple domain interactions. Moreover, we find that two non-overlapping protein fragments, one consisting of HRN/GTPase and the other consisting of HR1/HR2, can form a complex that reconstitutes Fzo1p fusion activity. Each of the null alleles disrupts the interaction of these two fragments, suggesting that we have identified a key interaction involving the GTPase domain and heptad repeats essential for fusion.The balance between fusion and fission maintains mitochondria as a dynamic tubular reticulum. In the absence of fusion, ongoing fission results in complete fragmentation of the mitochondrial network. Fragmentation leads to a loss of mtDNA and respiratory incompetence in yeast (1, 2) and accumulation of poorly functional mitochondria in mammals (3). Mice deficient for mitochondrial fusion die during mid gestation (4), and mutations in the mitochondrial fusion genes Mfn2 and OPA1 cause the neurodegenerative diseases Charcot-Marie-Tooth disease type 2A and dominant optic atrophy, respectively (5-9). Importantly, the mitochondrial fusion machinery is dedicated solely to mitochondrial fusion and does not include SNARE 2 proteins or NSF (N-ethylmaleimide-sensitive protein), which mediate most other intracellular membrane fusion events. Thus, mitochondrial fusion likely involves a unique mechanism.There are three essential components of the fusion pathway in yeast. The yeast mitofusin Fzo1p is a 98-kDa transmembrane GTPase uniformly distributed in the mitochondria outer membrane (1, 2). Both the N-and C-terminal portions of Fzo1p are oriented toward the cytosol, in position to mediate important steps during fusion (1, 2). Mgm1p is a dynamin-related protein in the mitochondrial intermembrane space (10 -12). It remains unclear if Mgm1p possesses the membrane severing activity of other dynamin family members, and if so, how this activity relates to its requirement for fusion. The third component of the fusion pathway is Ugo1p, a 58-kDa protein that spans the outer membrane once and interacts with both Fzo1p and Mgm1p (10,[12][13][14]. In this capacity, Ugo1p may anchor a fusion complex that coordinates the mitochondrial outer and inner membranes during fusion.Fzo1p is the best candidate to directly mediate fusion of the mitochondrial outer membrane. Unlike Mgm1p, it is positioned in the outer membrane and unlike Ugo1p its requisite ro...

Sequential requirements for the GTPase domain of the mitofusin Fzo1 and the ubiquitin ligase SCFMdm30 in mitochondrial outer membrane fusion

“…These findings, together with the known interaction between the N-and C-terminal halves of Fzo1 (Griffin and Chan, 2006), suggest a model in which an intact GTPase domain is required to relieve a steric block to Mdm30 binding imposed by the C-terminal HR1 and HR2-containing half of Fzo1.This model predicts that disruption of the structure of the C-terminal half of Fzo1, such that interactions with the N-terminal half of the molecule are interrupted, should result in increased Mdm30-mediated degradation. Consistent with this idea, mutations in either HR1 (L518P) or HR2 (L819P), which are each predicted to disrupt the structure of coiled-coils and abolish interactions with the N-terminal half of Fzo1 (Griffin and Chan, 2006), resulted in rapid degradation of Fzo1 (Fig. 2D, (Fig.…”

“…1A). When expressed alone, neither the cytosolic Myc-Fzo1-N nor the transmembrane HA-Fzo1-C fragments complement the absence of full-length Fzo1 in fzo1 cells, as assessed by growth on glycerol (Griffin and Chan, 2006). However, when expressed together, these two polypeptides form a functional complex at the mitochondrial outer membrane that rescues mitochondrial fusion and respiration (Griffin and Chan, 2006).…”

“…To further assess the relationship between the GTPase domain of Fzo1 and Mdm30-mediated ubiquitylation, we chose to evaluate the Fzo1 S201N GTPase mutant, which differs from other Fzo1 GTPase mutants as it is not complemented by co-expression of Fzo1 molecules with intact GTPase domains (Amiott et al, 2009;Griffin and Chan, 2006). As shown in Fig.…”

“…In yeast, an additional coiled-coil domain (HRN) is located N-terminal to the GTPase domain. Collectively the coiled-coil and GTPase domains are required to promote mitochondrial tethering and subsequent mitochondrial outer membrane (MOM) fusion (Griffin and Chan, 2006;Hermann et al, 1998;Ishihara et al, 2004;Koshiba et al, 2004). However, the sequence of events leading to fusion is poorly understood.…”

Structure, function, and regulation of mitofusin‐2 in health and disease

“…Outer membrane fusion is mediated by the mitofusins, large GTPases that traverse the outer mitochondrial membrane twice, with the amino and carboxy termini both facing into the cytoplasm (Alexander et al, 2000). Mitofusins form both homo-oligomeric (Mfn1-Mfn1 or Mfn2-Mfn2) and hetero-oligomeric (Mfn1-Mfn2) complexes in trans between apposing mitochondria (Chen et al, 2003;Griffin & Chan, 2006;Qi et al, 2016). Prior to fusion, curving of the outer membranes is promoted by the hydrolysis of cardiolipin to phosphatidic acid, a process mediated by phospholipase-D .…”