DLP1 Tubulates Lipid Membranes
Mammalian Dynamin-like Protein DLP1 Tubulates Membranes
Mol Biol Cell. 2001 September; 12 (9): 2894-2905
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-5‚Ä?-O-(3-thio)triphosphate (GTPŒ*S) but not GTP. EM of the GTPŒ*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 GTPŒ*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.
In living cells
DLP1-K38A aggregates are comprised of constricted ER membrane tubules with periodic DLP1 striations (in vivo).
Liposomes in vitro
Purified DLP1 tubulates lipid membranes in vitro. When DLP1 was incubated with liposomes prepared from either brain lipids or phosphatidylserine (PS), long membrane tubules were formed. Addition of GTPgS greatly enhanced the tubule formation, suggesting that the GTP-bound form of DLP1 more efficiently induces liposome tubulation. The morphology of tubules is similar to the ones formed with the conventional dynamin whereas branched tubules are frequently found in tubules prepared with DLP1.
In higher magnification, closely aligned striations on the tubule are visible. These striations are more apparent on the GTPgS-induced tubules. Striations are also frequently found on the surface of tubulating liposomes in the GTPgS-treated sample.
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