Our results show that the Net2 protein contains at least two functional domains, both playing important, but distinct, roles in mitochondrial division. Because the amino-terminal domain of Net2p interacts with Fis1p and the carboxyl-terminal region, Net2WDp, interacts with Dnm1p and because there is no evidence that Fis1p and Dnm1p directly interact (K. Cerveny, unpublished observations), it is tempting to speculate that Net2p links Fis1p and Dnm1p at sites of fission. However, our data suggest that Net2p is more than a simple bridging molecule. We show that the carboxyl-terminal WD-repeat region of Net2p interacts with both Dnm1p and Fis1p, and we have isolated mutations in the WD-repeat region of Net2p that primarily disrupt either Net2p-Dnm1p binding (Net2pR461A) or Net2p-Fis1p binding (Net2pF400A). Moreover, our data show that both of the mutated Net2 proteins are at least partially defective in mitochondrial division, indicating that the interaction of Net2p's WD-repeats with both Dnm1p and Fis1p is important for Net2p function.
Net2p is predicted to contain six or seven WD-40 repeats near its carboxyl-terminus. Structural studies have shown that the WD-40 repeats form seven blades, each consisting of four β-sheets connected by looped linker regions. These blades assemble into a barrel-shaped propeller structure that can act as a platform for protein-protein interactions (Wall et al., 1995
; Saxena et al., 1996
; Sondek et al., 1996
; Sprague et al., 2000
). In proteins such as Gβ1 and Tup1p, the top surface of the propeller contacts the partner proteins, with the loops between the β-strands providing much of the specificity (Garritsen and Simonds, 1994
; Wall et al., 1995
; Sondek et al., 1996
; Komachi and Johnson, 1997
; Sprague et al., 2000
; Zhang et al., 2002
). On the basis of our binding and localization analysis of the Net2p mutants, NET2F400A
, we propose that the WD-repeats of Net2p contact both Dnm1p and Fis1p on its top surface. The location of the analogous residues in the Tup1 structure, suggest that F400 and R461 may be close to each other (~10 angstroms), raising the possibility that the Fis1p and Dnm1p binding sites on Net2WDp overlap.
Dnm1p is a dynamin-related GTPase, and the Net2p-Dnm1p association appears to depend on the nucleotide state of Dnm1p. Unlike wild-type Dnm1p, GTPase mutant versions of Dnm1p no longer interact with the WD-repeats of Net2p in the yeast two-hybrid assay. Moreover, in cells expressing the altered Dnm1 proteins, Net2p is no longer associated with Dnm1p in dot-like structures on the mitochondria. Instead, Net2p is evenly dispersed along the mitochondrial surface, presumably bound to the Fis1 protein. This distribution of Net2p is identical to that seen in dnm1
Δ cells, arguing that the in vivo interaction of Net2p and Dnm1p is nucleotide dependent. Nearly all G-proteins undergo conformational changes upon GTP binding and hydrolysis, and many of these proteins such as Gα
, ras, and rabs have been shown to preferentially interact with their partners in a nucleotide-dependent manner (Sarvazyan et al., 1998
; Corbett and Alber, 2001
; Li et al., 2001
; Moyer et al., 2001
; Short et al., 2001
; Weide et al., 2001
). Although the S42N and T62F mutations correspond to those already analyzed in the homologous dynamin protein, it remains controversial whether dynamin's GTP binding, hydrolysis, or both are affected by these changes (Hinshaw and Schmid, 1995
; Binns et al., 1999
; Sever et al., 1999
; Damke et al., 2001
; Marks et al., 2001
; Eccleston et al., 2002
). Furthermore, it is unclear how the enzymatic activities of Dnm1p and dynamin compare. Consequently, studies are underway to biochemically characterize purified versions of Dnm1p, Dnm1pS42N
, and Dnm1pT62F
Based on the properties of dynamin and its similarity to the Dnm1 protein, there are at least two possible models that explain the roles that Dnm1p, Net2p, and Fis1p play in mitochondrial division. Dynamin has been shown to form collars around clathrin-coated plasma membrane invaginations to mediate membrane scission (Takei et al., 1995
). In one model for dynamin action, the assembly-stimulated GTP hydrolysis and the resulting conformational changes in the dynamin collar are proposed to constrict and pinch off the endocytic vesicle (Damke et al., 1994
; Hinshaw and Schmid, 1995
; Warnock et al., 1996
; Sweitzer and Hinshaw, 1998
). If Dnm1p acts in a similar manner, it could encircle mitochondria at specific sites and in a GTPase-dependent step, squeeze the mitochondrial tubule in two. In this scenario, Net2p and Fis1p would act primarily as scaffold proteins, anchoring Dnm1p to the mitochondria surface. In an alternative model, dynamin is proposed to function as a regulatory molecule that recruits and activates the actual membrane scission machinery in its GTP-bound state (Sever et al., 1999
; Fish et al., 2000
). If Dnm1p functions in this way, GTP-Dnm1p could either recruit factors, such as Net2p, to the site of scission, or activate proteins, like Fis1p, that may be already present.
Our data are most consistent with the idea that Dnm1p acts as a regulatory molecule, directly recruiting Net2p. For example, when the Net2p-Dnm1p interaction is disrupted, by either a mutation in the WD-repeats of Net2p or a defect in the GTPase region of Dnm1p, the distribution of Net2p changes dramatically. Instead of being in dot-like structures, the Net2 protein in cells expressing Net2pR461A, Dnm1pS42N, or Dnm1pT62F is found all along the mitochondrial surface. Dnm1p in these cells remains punctate. These observations argue that the location of Net2p is dependent on Dnm1p, but Dnm1p's distribution does not strictly require Net2p. Furthermore, if Net2p functions as a simple adapter molecule, bringing soluble Dnm1p together with the membrane-bound Fis1 protein, then excess Net2Np or Net2WDp would interfere with Net2p-Fis1p or Net2p-Dnm1p binding and cause release of Dnm1p from mitochondria. However, in cells overproducing Net2Np or Net2WDp, Dnm1p remains associated with mitochondria. Perhaps the strongest evidence that Dnm1p recruits Net2p is provided by our GAL1-NET2 induction studies. Net2p has two potential binding sites on mitochondria: either Fis1p, which is evenly distributed all along the outer membrane, or Dnm1p, which is located in discrete patches. When the expression of the Net2 protein is induced in cells, Net2p first appears in dot-like structures on mitochondria. Only when the Dnm1p-Net2p interaction is disrupted do we see Net2p all along the mitochondrial tubule bound to Fis1p. Together, our observations suggest that Dnm1p acts early in the division pathway and functions to recruit effector proteins, such as Net2p and Fis1p. Consistent with this idea, we find that Dnm1p can bind to specific sites on the mitochondria in the absence of either Fis1p or Net2p.
In a recent report, Tieu et al.
) suggest that Net2p functions as an adapter molecule, bringing together Fis1p and Dnm1p. Their data indicate that the amino-terminal region of Net2p interacts with Fis1p, whereas the carboxyl-terminal region binds only Dnm1p. They also showed that expression of either the amino- or carboxyl-terminal domains of Net2p block mitochondrial division. We confirmed and extended their studies by showing that Net2WDp was able to bind Dnm1p and, to a lesser extent, Fis1p. In addition, we identified key residues in the carboxyl-terminal WD-repeats of Net2p and the GTPase domain of Dnm1p that are required for assembly of the known mitochondrial fission components. Furthermore, we found that a direct physical interaction between Dnm1p and the WD-repeats of Net2p was responsible for the dominant inhibition of mitochondrial scission caused by Net2WDp. Tieu et al.
) proposed a model in which Fis1p recruits first Dnm1p and then Net2p to the site of mitochondrial division. However, because Fis1p is evenly distributed along the mitochondrial surface, it is difficult to imagine how Fis1p can recruit Dnm1p to a specific site. We suggest a different scenario, in which Dnm1p marks the site of fission and then recruits effector molecules, such as Net2p and Fis1p. Supporting this view, we see that much of Dnm1p remains on the mitochondria and in punctate spots in the absence of Fis1p. We speculate that some unknown protein or lipid modification is initially recognized by Dnm1p. Furthermore, although the precise role of Net2p in fission is not known, we suggest that at least one of the functions of Net2p is to coordinate the activities of Dnm1p and Fis1p by organizing the division complex. Consistent with this idea, we find that mutations in the GTPase region of Dnm1p, defects in the WD repeat-containing portion of Net2p, or the lack of Fis1p all seem to block mitochondrial fission at a common step. In all cases, Dnm1p is found in several large aggregates on the mitochondrial surface, and the cytosolic pool of Dnm1p appears to be absent. Additionally, if Dnm1p is artificially assembled (e.g., by fusion of dsRed to its carboxyl-terminus), mitochondrial scission is blocked and the dominant-negative Dnm1p-dsRed protein is found in large aggregates similar to those seen in Dnm1p and Net2p mutant cells (K. Cerveny, unpublished observations). Thus, the normal cycle of the Dnm1 protein appears to require productive and regulated interactions with Net2p, Fis1p, and Dnm1p, with Net2p playing a pivotal role in their coordination. Studies in progress are aimed at understanding the GTPase cycle of Dnm1p in the context of Net2p, Fis1p, and other potential mitochondrial fission proteins.