Mammalian dynamin-like protein DLP1 tubulates membranes

Dynamins are large GTPases with mechanochemical properties that axe known t o constrict and tabulate membranes. A recently identified mammalian dynamin -like protein (DLP1) is essential for the proper cellular distribution of m itochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conven tional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggr egates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed tha t these aggregates were comprised of membrane tubules of a consistent diame ter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the a ssociation of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds b ut does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38 A for membrane is increased compared with wild-type DLP1. To test whether D LP1 could tabulate membrane in vitro, recombinant DLP1 was combined with sy nthetic liposomes and nucleotides. We found that DLP1 protein alone assembl ed into sedimentable macromolecular structures in the presence of guanosine -5 ' -O-(3-thio)triphosphate (GTP gammaS) but not GTP. EM of the GTP gammaS -treated DLP1 revealed clusters of stacked helical ring structures. When li posomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTP gammaS great ly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP 1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These resul ts provide the first evidence that the dynamin family member, DLP1, is able to tabulate membranes both in living cells and in vitro. Furthermore, thes e findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.