Abstract
Dnm1p/Vps1p-like protein (DVLP) is a mammalian member of the dynamin GTPase family, which is classified into subfamilies on the basis of the structural similarity. Mammalian dynamins constitute the dynamin subfamily. DVLP belongs to the Vps1 subfamily, which also includes yeast Vps1p and Dnm1p. Typical structural features that discriminate between members of the Vps1 and dynamin subfamilies are that the former lacks the pleckstrin homology and Pro-rich domains. Dynamin exists as tetramers under physiological salt conditions, whereas under low salt conditions, it can polymerize into spirals that resemble the collar structures seen at the necks of constricted coated pits. In this study, we found that DVLP is also oligomeric, probably tetrameric, under physiological salt conditions and forms sedimentable large aggregates under low salt conditions. The data indicate that neither the pleckstrin homology nor Pro-rich domain is required for the self-assembly. Analyses using the two-hybrid system and co-immunoprecipitation show that the N-terminal region containing the GTPase domain and a domain (DVH1) conserved across members of the dynamin and Vps1 subfamilies, can interact with the C-terminal region containing another conserved domain (DVH2). The data on the interdomain interaction of DVLP is compatible with the previous reports on the interdomain interaction of dynamin. Thus, the self-assembly mechanism of DVLP appears to resemble that of dynamin, suggesting that DVLP may also be involved in the formation of transport vesicles.
Highlights
Dynamin is a high molecular weight GTP-binding protein with an intrinsic GTPase activity
Typical structural features that discriminate between members of the Vps1 and dynamin subfamilies are that the former lack the pleckstrin homology and Pro-rich domains, which are involved in interactions with phospholipids and proteins containing the SH3 domain, respectively
Yoon et al [7] showed by immunoelectron microscopy that DLP1/Dnm1p/Vps1p-like protein (DVLP)-positive structures coalign with microtubules and tubules of the endoplasmic reticulum and proposed that DLP1/DVLP may participate in the formation of nascent secretory vesicles from the endoplasmic reticulum
Summary
Plasmid Construction—Construction of the expression vector for human DVLP with an N-terminal hemagglutinin (HA) epitope sequence (pcDNA3-HAN-DVLP) was described previously [5]. For the use in the two-hybrid analysis, bait and prey vectors for DVLP were constructed by ligation of a DNA fragment for DVLP from pcDNA3-HAN-DVLP into pGBT9 and pGAD10 (CLONTECH), respectively. The mixture was electrophoresed on a 7% SDSpolyacrylamide gel under nonreducing conditions and subjected to Western blot analysis with anti-HA antibody as described above. A half of the lysates containing ϳ20 g of protein were directly subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis using anti-HA (3F10) or anti-Myc (9E10) antibody. The other half was immunoprecipitated with anti-HA antibody (12CA5) and protein A-Sepharose and subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis using the monoclonal anti-Myc antibody. The reaction product was immunoprecipitated with anti-HA antibody (12CA5) and protein A-Sepharose, electrophoresed on SDS-polyacrylamide gel, and analyzed using a BAS2000 bioimaging analyzer (Fuji Film Co.)
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