Dymple, a Novel Dynamin-like High Molecular Weight GTPase Lacking a Proline-rich Carboxyl-terminal Domain in Mammalian Cells

Abstract

We have cloned human dymple, a novel dynamin family member. The full-length cDNA sequence encodes a protein composed of 736 amino acids with a molecular mass of 80 kDa. This amino acid sequence most resembles yeast DNM1P and VPS1P. Dymple lacks a prolinerich carboxyl-terminal domain through which dynamin binds to SH3 domains to be activated. Northern blot analysis revealed two transcript sizes of 2.5 and 4.2 kilobases with alternative polyadenylation at the highest levels in brain, skeletal muscle, and testis. It was further established that there are three patterns of alternative splicing producing in-frame deletions in the coding sequence of dymple in a tissue-specific manner. When overexpressed, wild-type dymple exhibited a punctate perinuclear cytoplasmic pattern, whereas an amino-terminal deletion mutant formed large aggregates bounded by a trans-Golgi network marker. Since dynamin participates in clathrin-mediated endocytosis through a wellcharacterized mechanism, the existence of a dynaminlike molecule in each specific vesicle transport pathway has been predicted. Our findings suggest that dymple may be the first example of such a subfamily in mammalian cells other than dynamin itself, although its precise role and membrane localization remain to be resolved.

Spectrum of autoantibodies against a dynamin-related protein, dymple

“…Serum from a patient with SSc (patient KK) was used to screen a HeLa Zap cDNA expression library, and several positive clones were identified as dymple clones (2). Clone D104 encodes full-length dymple protein, and clone N246 derived from clone D104 encodes amino acids 246-736 of dymple (Figure 1).…”

“…The amino-terminal deletion mutant N246 was created by the deletion of the Hinc II fragment (1-245 amino acids) of the dymple insert in pBluescript II SK(Ϫ) (2) and subcloned as a Xho I-Not I fragment into the Sal I-Not I site of the pGEX 5X-3 vector. Escherichia coli JM109 cells harboring each pGEX-derived plasmid were grown, and the expression of the GST-fused proteins was induced as described previously (2). To purify GST-fusion protein, glutathione-Sepharose 4B (Pharmacia) was used according to the manufacturer's protocol.…”

“…To express the recombinant protein as a GST fusion protein, clone D104, which encodes the full-length protein, was constructed with the bacterial expression vector pGEX 5X-3 (Pharmacia, Uppsala, Sweden) as in our previous study (2). The amino-terminal deletion mutant N246 was created by the deletion of the Hinc II fragment (1-245 amino acids) of the dymple insert in pBluescript II SK(Ϫ) (2) and subcloned as a Xho I-Not I fragment into the Sal I-Not I site of the pGEX 5X-3 vector.…”

“…These proteins have a highly conserved N-terminal GTPase domain of ϳ300 amino acids (3). Dymple belongs to a subfamily whose members lack the pleckstrin homology and proline-rich domains contained in dynamin subfamilies (2). Dymple has also been described by other groups of investigators, who have used other names, including DVLP (Dnm1p/Vps1p-like protein) (4), DLP1 (dynamin-like protein 1) (5), and DRP1 (dynaminrelated protein 1) (6).…”

“…Recently, we cloned a novel antigen, dymple, a member of the dynamin family that lacks a proline-rich carboxyl-terminal domain, using serum from a patient with scleroderma (2). Dynamin is a high molecular weight GTP-binding protein with intrinsic GTPase activity.…”