Cytokinesis failure in clathrin-minus cells is caused by cleavage furrow instability.

The role of membrane traffic during cell division has only recently begun to be investigated. A growing number of trafficking proteins seem to be involved in the successful completion of cytokinesis. Clathrin was the first trafficking protein to be shown to be essential for cytokinesis in Dictyostelium. Here we investigate the nature of the cytokinesis defect of Dictyostelium clathrin null cells. We found that adherent clathrin null cells do form cleavage furrows but cannot maintain a consistent rate of furrow ingression. Clathrin null cells are completely defective in cytokinesis when placed in suspension. In these conditions, the cells develop an abnormal division morphology that consists of two lateral "furrows" on either side of a bulging equatorial region. Cells expressing GFP-myosin II were examined at various stages of cytokinesis. Clathrin null cells show multiple defects in myosin organization and localization that parallel the striking failure in furrow morphology. We postulate that this morphology is the result of contraction at the rear of the presumptive daughter cells in concert with incomplete furrow ingression.

Spatially regulated recruitment of clathrin to the plasma membrane during capping and cell translocation.

Clathrin-coated vesicles bud from selected cellular membranes to traffic-specific intracellular proteins. To study the dynamic properties of clathrin-coated membranes, we expressed clathrin heavy chain tagged with green fluorescent protein (GFP) in Dictyostelium cells. GFP-clathrin was functional and retained the native properties of clathrin: the chimeric protein formed classic clathrin lattices on cellular membranes and also rescued phenotypic defects of clathrin null cells. GFP-clathrin distributed into punctate loci found throughout the cytoplasm, on the plasma membrane, and concentrated to a perinuclear location. These clathrin-coated structures were remarkably motile and capable of rapid and bidirectional transport across the cell. We identified two local domains of the plasma membrane as sites for clathrin recruitment in motile cells. First, as cells translocated or changed shape and retracted their tails, clathrin was transiently concentrated on the membrane at the back of the cell tail. Second, as cells capped their cell surface receptors, clathrin was recruited locally to the membrane under the tight cap of cross-linked receptors. This suggests that local sites for clathrin polymerization on specific domains of the plasma membrane undergo rapid and dynamic regulation in motile cells.

Rapid identification of protein phosphatase 1-binding proteins by mixed peptide sequencing and data base searching. Characterization of a novel holoenzymic form of protein phosphatase 1.

Microcystin-affinity chromatography was used to purify 15 protein phosphatase 1 (PP1)-binding proteins from the myofibrillar fraction of rabbit skeletal muscle. To reduce the time and amount of material required to identify these proteins, proteome analysis by mixed peptide sequencing was developed. Proteins are resolved by SDS-polyacrylamide gel electrophoresis, electroblotted to polyvinylidene fluoride membrane, and stained. Bands are sliced from the membrane, cleaved briefly with CnBr, and applied without further purification to an automated Edman sequencer. The mixed peptide sequences generated are sorted and matched against the GenBank using two new programs, FASTF and TFASTF. This technology offers a simple alternative to mass spectrometry for the subpicomolar identification of proteins in polyacrylamide gels. Using this technology, all 15 proteins recovered in PP-1C affinity chromatography were sequenced. One of the proteins, PP-1bp55, was homologous to human myosin phosphatase, MYPT2. A second, PP-1bp80, identified in the EST data bases, contained a putative PP-1C binding site and a nucleotide binding motif. Further affinity purification over ATP-Sepharose isolated PP-1bp80 in a quaternary complex with PP-1C and two other proteins, PP-1bp29 and human p20. Recombinant PP-1bp80 also bound PP-1C and suppressed its activity toward a variety of substrates, suggesting that the protein is a novel regulatory subunit of PP-1.

Calcium-dependent self-association of synaptotagmin I.

Synaptotagmin I, an integral membrane protein of secretory vesicles, appears to have an essential role in calcium-triggered hormone and neurotransmitter release. The large cytoplasmic domain of synaptotagmin I has two C2 domains that are thought to mediate calcium and phospholipid binding. A recombinant protein (p65 1-5) comprised of the cytoplasmic domain was previously shown to aggregate purified chromaffin granules and artificial phospholipid vesicles in a calcium-dependent manner. p65 1-5 may be able to aggregate membrane vesicles by a self-association reaction. This hypothesis led us to investigate the ability of synaptotagmin I protein fragments to multimerize in vitro. We found that p65 1-5, in the absence of membranes, was able to self-associate to form large aggregates in a calcium-dependent manner as shown by light-scattering assays and electron microscopy. In addition, a recombinant protein comprised of only the second half of the cytoplasmic domain, including the second C2 domain, was also able to self-associate and aggregate phospholipid vesicles in a calcium-dependent manner. A recombinant protein comprised of only the first C2 domain was not able to self-associate or aggregate vesicles. These results suggest that synaptotagmin I is able to bind calcium in the absence of membranes and that the second half of the cytoplasmic domain is able to bind calcium and mediate its multimerization in a calcium-dependent manner. The ability of synaptotagmin I protein fragments to multimerize in a calcium-dependent manner in vitro suggests that multimerization may have an important function in vivo.

Synaptotagmin II expression partially rescues the growth defect of the yeast sec15 secretory mutant.

Synaptotagmins are a family of calcium- and phospholipid-binding proteins implicated in the function of cell exocytosis. Synaptotagmins I and II are neurally expressed proteins thought to be involved in neurotransmitter release from neurons. We have expressed rat synaptotagmin II in several Saccharomyces cerevisiae temperature-sensitive secretory mutants that are defective in Golgi to plasma membrane vesicular transport. Synaptotagmin II expression was able to partially rescue the growth defect in one particular mutant, sec15. No suppression was observed when synaptotagmin II was expressed in sec1, sec2, sec4, sec5, sec6, sec8, sec9, sec14, sec17, or sec18. Two synaptotagmin II deletion mutants were also expressed in sec15 and screened for suppression. The expression of the cytoplasmic domain of synaptotagmin alone was not able to suppress the sec15 growth defect. In addition, the expression of a synaptotagmin II fragment lacking the second half of the cytoplasmic domain including the second C2 domain did not suppress sec15. We have isolated a membrane fraction enriched in post-Golgi vesicles from a sec15 strain expressing synaptotagmin II and found that synaptotagmin II co-purifies with this fraction, suggesting that the rat synaptotagmin II is targeted to membranes in yeast. Sec15p forms a large multisubunit protein complex that includes Sec6p and Sec8p. This protein complex is thought to function in a late stage of exocytosis in yeast. Sec6p and Sec8p homologs have been identified in mammalian cells. Our studies suggest that synaptotagmin may be a part of this complex or regulate its function in mammalian cells.

Synergistic membrane interactions of the two C2 domains of synaptotagmin.

Synaptotagmin, an integral membrane protein localized to secretory vesicles, has been implicated in the docking and fusion steps in calcium-regulated exocytosis. The large cytoplasmic domain contains two C2 motifs, each similar to the Ca2+ and phospholipid binding domain of protein kinase C. To study the membrane binding and aggregating properties of these C2 domains, three recombinant fragments of rat synaptotagmin I were expressed in Escherichia coli and purified. A recombinant protein containing both C2 domains (p65 1-5) was found to bind to and aggregate bovine chromaffin granules in a calcium-dependent manner, with half-maximal binding and aggregation occurring at approximately p Ca2+ = 4.2. However, recombinant proteins containing either the first (p65 1-3) or second (p65 3-5) C2 domain alone were not able to bind to the granules, indicating that both C2 domains are required for binding to chromaffin granules. p65 1-5 also bound to and aggregated liposomes made from chromaffin granule lipid extracts, as well as granules treated extensively with trypsin, suggesting that p65 1-5 binding to granules is mediated by the lipids in the granule membrane and not the granule membrane proteins. Although p65 1-3 and p65 3-5 did not bind to granules or lipids extracted from granules, both did bind to phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (10%-40%PS). Half-maximal binding of p65 1-3 to vesicles occurred at approximately p Ca2+ = 5.2, while p65 3-5 appeared to bind independently of calcium over the range of pCa2+ = 5.5-2.8. p65 1-5 exhibited binding to PS/PC vesicles with characteristics of both the smaller proteins, displaying some binding in EGTA and increased binding in calcium. Larger amounts of p65 1-5 bound to PS/PC vesicles than of either of the smaller fragments. These results suggest that the two C2 domains of synaptotagmin act synergistically to promote binding to biological membranes and to affect calcium sensitivity and membrane binding capacity.