Dynamin Interacts with Members of the Sumoylation Machinery

Abstract

Dynamin is a GTP-binding protein whose oligomerization-dependent assembly around the necks of lipid vesicles mediates their scission from parent membranes. Dynamin is thus directly involved in the regulation of endocytosis. Sumoylation is a post-translational protein modification whereby the ubiquitin-like modifier Sumo is covalently attached to lysine residues on target proteins by a process requiring the concerted action of an activating enzyme (ubiquitin-activating enzyme), a conjugating enzyme (ubiquitin carrier protein), and a ligating enzyme (ubiquitin-protein isopeptide ligase). Here, we show that dynamin interacts with Sumo-1, Ubc9, and PIAS-1, all of which are members of the sumoylation machinery. Ubc9 and PIAS-1 are known ubiquitin carrier protein and ubiquitin-protein isopeptide ligase enzymes, respectively, for the process of sumoylation. We have identified the coiled-coil GTPase effector domain (GED) of dynamin as the site on dynamin that interacts with Sumo-1, Ubc9, and PIAS-1. Although we saw no evidence of covalent Sumo-1 attachment to dynamin, Sumo-1 and Ubc9 are shown here to inhibit the lipid-dependent oligomerization of dynamin. Expression of Sumo-1 and Ubc9 in mammalian cells down-regulated the dynamin-mediated endocytosis of transferrin, whereas dynamin-independent fluid-phase uptake was not affected. Furthermore, using high resolution NMR spectroscopy, we have identified amino acid residues on Sumo-1 that directly interact with the GED of dynamin. The results suggest that the GED of dynamin may serve as a scaffold that concentrates the sumoylation machinery in the vicinity of potential acceptor proteins.The protein dynamin is an important component of the endocytic machinery in cells (1, 2). Dynamin isoforms are thought to mediate their functions by virtue of their ability to aid in the scission of vesicles from membranes (3). Mammalian dynamin-1 and dynamin-2 have been shown to have a role in the scission of clathrin-coated vesicles and in the budding of caveolae (4). The dynamin family can be considered to include classical dynamins as well as the dynamin-like mitochondrial division proteins Dnm1, Mgm1, and Fzo1 and the interferoninducible Mx and guanylate-binding proteins, which have been shown to possess antiviral activities (5). The yeast Vps1 and plant ARC5 and ADL proteins also belong to this family of GTP-binding proteins (6). True dynamins are modular proteins characterized by the presence of an N-terminal GTP-binding domain, a contiguous "middle domain" of ill defined function, and a lipid-binding pleckstrin homology (PH) 1 domain, followed by a coiled-coil "assembly" domain and a proline-rich domain (PRD). Other dynamin-like proteins, e.g. Mx, lack the pleckstrin homology and proline-rich domains. The GTPase domain is the most highly conserved domain within members of the dynamin family. The coiled-coil assembly domain has been shown to mediate the assembly of dynamin into oligomers (7) and has also been shown to possess an assembly-stimulated GTPase-accelerating property for th...

Small Ubiquitin-like Modifier (SUMO) Recognition of a SUMO Binding Motif

“…These sequences are similar to that of the SUMO-1 binding site of PIASX (VDVIDLT), determined by our previous study (22). There is also the possibility that other SUMO binding sequences may exist (26,27).…”

“…These sequences are similar to that of the SUMO-1 binding site of PIASX (VDVIDLT), determined by our previous study (22). There is also the possibility that other SUMO binding sequences may exist (26,27).The physiological relevance has been demonstrated for the SBM that we have previously identified (28). An investigation combining sitedirected mutagenesis of SUMO-1 and SUMO-2 and transcriptional assays have identified a specific surface of SUMO as the only region that mediates SUMO-1 and -2 dependent transcriptional inhibition.…”

Pias1 Interaction and Sumoylation of Metabotropic Glutamate Receptor 8

“…Furthermore, overexpression of the sumoylation cascade in mammalian cells can down-regulate the dynamin-mediated endocytosis of other proteins, e.g. transferrin (45). It therefore is tempting to speculate that dynamin serves to localize the primarily nuclear sumoylation machinery in the vicinity of target plasma membrane proteins.…”

“…There is circumstantial evidence that sumoylation can happen at the plasma membrane. Dynamin has been shown to interact with ubc9, sumo1, and Pias1 without being sumoylated itself (45), and to be involved in internalization of the group III mGluR4 (46). Furthermore, overexpression of the sumoylation cascade in mammalian cells can down-regulate the dynamin-mediated endocytosis of other proteins, e.g.…”

Dynamin Functions and Ligands: Classical Mechanisms Behind

“…Reversible phosphorylation of human dynamin-related protein 1 (DRP1) at synaptic vesicles occurs via calcium/calmodulin-dependent protein kinase, cyclin-dependent kinase 1, and protein kinase A. Apart from phosphorylation, DRP1 has been shown to undergo SUMOylation, which increases mitochondrial fission (Mishra et al, 2004).…”