Mammalian Dynamin-like Protein DLP1 Tubulates Membranes

Abstract

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5Ј-O-(3-thio)triphosphate (GTP␥S) but not GTP. EM of the GTP␥S-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTP␥S greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.

The GTPase Effector Domain Sequence of the Dnm1p GTPase Regulates Self-Assembly and Controls a Rate-limiting Step in Mitochondrial Fission

“…Although it is unclear why we have not observed similar Dnm1p rings/collars in vitro, one possibility is that one or more components of the Dnm1p-mediated fission machinery (e.g., Mdv1p and/or Fis1p) are required for the formation of functional, higher order Dnm1p structures (Mozdy et al, 2000;Tieu and Nunnari, 2000). Based on the observation that the Dnm1p homolog DRP1/DLP1 can form oligomers (Yoon et al, 2001), and our in vivo evidence that Dnm1p is required for mitochondrial fission, we think it is likely that Dnm1p also forms rings or collars around mitochondrial tubules at sites where constriction and fission will occur.…”

“…Recently, the Caenorhabditis elegans (DRP-1) (Labrousse et al, 1999) and mammalian (DRP1/DLP1/DVLP) homologs of yeast Dnm1p were also shown to be required for mitochondrial fission in vivo (Smirnova et al, 2001). Bacterially expressed mammalian DRP1/DLP1 appeared to form stacked rings/collars that tubulated and constricted tubules composed of synthetic lipids (Yoon et al, 2001). Although it is unclear why we have not observed similar Dnm1p rings/collars in vitro, one possibility is that one or more components of the Dnm1p-mediated fission machinery (e.g., Mdv1p and/or Fis1p) are required for the formation of functional, higher order Dnm1p structures (Mozdy et al, 2000;Tieu and Nunnari, 2000).…”

Mitochondrial dynamics and division in budding yeast

“…Although Dnm1p is related to dynamin, there is currently no ultrastructural evidence that this yeast protein assembles into higher-order structures. However, two recent studies showed that mammalian Drp1 (also called Dlp1) [26,29] assembles to form rings in vitro.…”

“…This ring formation induces tubulation of synthetic liposomes, raising the possibility that the mitochondrial dynamin family members form rings around mitochondrial membranes in vivo before organelle fission [29]. Clearly, more efforts must be made to determine whether Dnm1p forms rings and whether the rings formed in vitro are functionally and structurally equivalent to the punctate structures that regulate mitochondrial fission in vivo.…”

The many shapes of mitochondrial death

“…This suggests that all dynamin-related fission molecules may function by a common mechanism (Praefcke and McMahon, 2004). Mammalian Drp-1 has also been shown to form tubules (Yoon et al, 2001), but other molecules are required to organize and constrict mitochondria after formation of Drp-1 tubules. In particular, knocking down endophilin B1, a member of a family of proteins involved in endocytic vesicle formation, promotes the formation of Drp-1 tubules that elongate through the cytoplasm without breaking down the mitochondrial network (Karbowski et al, 2004b).…”