Division of mitochondrial tubules contributes to the overall morphology of the mitochondrial reticulum (Yaffe 1999a
). Our findings reveal that the gag
mutations define components that mediate mitochondrial fission. gag1
mapped to DNM1
that previously was shown to encode a dynamin-related protein essential for mitochondrial division (Otsuga et al. 1998
; Bleazard et al. 1999
; Sesaki and Jensen 1999
). Three observations indicate that GAG2
also encode fission factors. First, the gag2
mutants display defects in mitochondrial morphology that are essentially identical to those observed in dnm1
mutant cells (). Second, as previously shown for DNM1
, mutations in both GAG2
prevent mitochondrial fragmentation caused by loss of the fusion factor, Fzo1p ( A). Finally, mitochondria in gag2
, and dnm1
mutants remain tubular upon addition of sodium azide, an agent that stimulates the fragmentation of mitochondrial tubules in wild-type cells ( C).
Both Gag3p and Dnm1p function on the outer membrane to mediate mitochondrial division, yet they display distinct characteristics in their association with the mitochondrial surface. Gag3p is tightly bound to the mitochondrial outer membrane, whereas the association of Dnm1p appears weaker, since the protein is recovered with the cytosolic fraction during subcellular fractionation ( A; Otsuga et al. 1998
). Additionally, the gag2
mutation causes displacement of a major fraction of Dnm1p to the cytosol in intact cells (), yet Gag3p remains tightly bound to mitochondria isolated from gag2
mutant cells (data not shown). Although two-hybrid analysis and localization studies are consistent with an interaction of Dnm1p and Gag3p, such an association is not required for the localization of the two proteins to the outer membrane, since neither protein is displaced in the absence of the other. These differences suggest that Gag3p and Dnm1p play distinct roles in the fission process.
The specific molecular function of Gag3p in mitochondrial division is unknown. Unlike Dnm1p, whose homologue dynamin mediates the scission step at the neck of coated pits in animal cells (Schmid et al. 1998
, van der Bliek 1999
), no proteins related to Gag3p have been implicated in other membrane fission events. One clue to the protein's function might lie in its two distinct structural domains, a coiled-coil region in the NH2
-terminal half of the protein and six WD40-repeats in the COOH-terminal half. These features are likely to comprise protein interaction domains (Smith et al. 1999
) and suggest a role for Gag3p in bringing together or organizing multiple components on the mitochondrial surface. One interacting partner is likely to be Dnm1p, although the dissimilar behavior of these components during subcellular fractionation and our inability to detect binding with coimmunoprecipitation analysis (data not shown) suggest a transient or unstable interaction of these proteins. Gag3p is likely to bind other outer membrane proteins and, in particular, one or more integral membrane proteins. The identification of these binding partners and an analysis of the dynamic interactions of fission components will lead to the elucidation of molecular mechanisms that mediate mitochondrial division.