Dynamin is a GTPase that plays a critical role in the very early stages of endocytosis, regulating the scission of clathrin-coated and non-clathrin-coated pits from the plasma membrane. While the ligands through which dynamin exerts its in vivo effects are unknown, dynamin exhibits in vitro binding to several proteins containing Src homology 3 (SH3) domains, as well as to microtubules and anionic phospholipids, via a basic, prolinerich C-terminal domain. To begin to identify the in vivo binding partners of dynamin, we have examined by immunofluorescence the association of mutant and wildtype forms of dynamin with plasma membranes prepared by sonication of transiently transfected cells. Wild-type dynamin was found almost exclusively in association with clathrin-containing domains. Binding to these regions was abolished by removal of a nine-amino acid sequence within the C-terminal domain encoding a candidate SH3 domain binding site. Binding did not require clathrin and resisted extraction at both high and low ionic strength, consistent with an interaction with an SH3 domain. Surprisingly, we also find that dynamin contains multiple regions involved in binding to nonclathrin-containing domains, including a 13-amino acid sequence directly upstream of the C-terminal domain. These observations suggest that a protein containing an SH3 domain is involved in recruiting dynamin to coated pits and provide the first evidence for a biological role for SH3 domains in dynamin function.

Dynamin is a 100-kDa GTPase that plays a critical role in the initial stages of endocytosis. Dynamin binds to microtubules, which potentiy stimulate its GTPase activity.Binding to Src homology 3 (SH3) domains of proteins involved in signal transduction has also recently been reported. In the present study, the protein was digested with a variety of proteases to derme its functional domains. Limited digestion with papain split the protein into an -7-to 9-kDa microtubulebinding fragment and a 90-kDa nonbinding fragment. Immunoblotting with an antibody to the C-terminal 20 amino acids of rat dynamin showed the small fragment to derive from the C-terminal end ofthe polypeptide. Microtubule-activated GTPase activity, but not basal GTPase activity, was abolished by papain digestion, identifying the basic, proline-rich C-terminal region of dynamin as an important regulatory site. Bacterially expressed growth factor receptor-bound protein 2 (GRB2) and the SH3 domain of c-Src were also found to stimulate GTPase activity, although to a lesser extent than microtubules. Stimulation of GTPase activity by the recombinant proteins was similarly abolished by papain digestion. These results identify the basic, proline-rich C-terminal region of dynamin as the binding site for both microtubules and SH3 domains and demonstrate an allosteric interaction between this region of the molecule and the N-terminal GTPase domain.Dynamin was initially identified as a 100-kDa nucleotidesensitive microtubule-binding protein (1). In the absence of GTP, dynamin bundled microtubules, forming highly cooperative helical arrays along the microtubule surface (1). The deduced primary sequence of the rat brain protein (2) revealed an N-terminal 300-amino acid domain that contained the three highly conserved consensus-sequence elements characteristic of GTPases. This region of the molecule showed striking sequence similarity to the N-terminal regions of the interferon-inducible antiviral Mx proteins (3) and the product of the yeast vacuolar protein-sorting gene VPSI involved in membrane protein sorting (4) and meiosis (5). The dynamin sequence was found to diverge from VPS1 and Mx beyond the GTPase domain and, in addition, to contain a non-conserved 100-amino acid basic, proline-rich extension.The Drosophila gene shibire has been identified as a potential homologue of dynamin, exhibiting 68% amino acid sequence identity with the rat protein and containing a C-terminal extension of comparable length and composition (6, 7). Temperature-sensitive alleles of shibire have a paralytic phenotype (8) that has been attributed to a defect in the reformation of synaptic vesicles at the neuromuscular junction (9, 10) and, more generally, in the budding of coated and noncoated vesicles from the plasma membrane (11,12 The present study was initiated to gain further insight into the functional organization of the dynamin molecule and to determine whether SH3 domains interact with the same region within the molecule as microtubules. We report here that proteoly...

The GTPase dynamin has been implicated in the regulation of the scission of coated and noncoated pits during the early stages of endocytosis. Various macromolecules including microtubules, acidic phospholipids, and Src homology 3 (SH3) domains have been shown to interact with the basic, proline-rich region of dynamin and act as effectors of its GTPase activity. The interaction of dynamin with SH3 domain-containing proteins is of particular interest since SH3 domains are known to mediate protein-protein interactions in signal transducing complexes. In this study, we have systematically defined three distinct SH3 binding regions within the dynamin proline-rich C terminus. These binding regions conform to either the Class I or II SH3 binding consensus sequence, and their location coincides with a region previously shown to be important in the colocalization of dynamin with clathrin-coated pits. Two of these SH3 binding regions are well conserved among four dynamin isoforms, and we show that the overall binding pattern for SH3 domains is comparable among the isoforms. We also demonstrate that neither transferrin nor plateletderived growth factor receptor uptake is restored upon removal of the basic, proline-rich region in a dominant negative dynamin GTP binding mutant. Together with earlier evidence from our laboratory, these findings suggest that SH3 domains may serve to target dynamin to coated pits and are not the direct targets of dominant inhibitory mutants of dynamin.

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Cytoplasmic dynein is a multi-subunit complex involved in retrograde organelle transport and some aspects of mitosis. In previous work we have cloned and sequenced cDNAs encoding the rat cytoplasmic dynein heavy and intermediate chains. Here we report the cloning of the remaining class of cytoplasmic dynein subunits, which we refer to as the light intermediate chains (LICs: 53–59 kDa). Four LIC electrophoretic bands were resolved in purified bovine cytoplasmic dynein preparations by one-dimensional gel electrophoresis. These four bands were simplified to two bands (LIC53/55 and LIC57/59) by alkaline phosphatase treatment. N-terminal amino acid sequence was obtained from a total of 11 proteolytic peptides generated from both LIC53/55 and LIC57/59. Overlapping cDNA clones encoding LIC53/55 were isolated by oligonucleotide screening using probes based on the LIC53/55 peptide sequence. The cDNA sequence contained a 497 codon open reading frame encoding a polypeptide with a molecular mass of approximately 55 kDa. Each of the LIC53/55 peptides was found within the deduced amino acid sequence, as well as four of the LIC57/59 peptides. Analysis of the LIC53/55 primary sequence revealed homology with the ABC transporter family of ATPases in the region surrounding the P-loop sequence element. Together these data identify the LICs as a novel family of dynein subunits with potential ATPase activity. They also reveal that the complexity of the LICs is due to both post-translational modification and the existence of at least two LIC polypeptides for which we propose the names LIC-1a and LIC-2.