Copines make up a family of soluble, calcium-dependent, membrane binding proteins found in a variety of eukaryotic organisms. In an earlier study, we identified six copine genes in the Dictyostelium discoideum genome and focused our studies on cpnA. Our previous localization studies of green fluorescent protein-tagged CpnA in Dictyostelium suggested that CpnA may have roles in contractile vacuole function, endolysosomal trafficking, and development. To test these hypotheses, we created a cpnA ؊ knockout strain, and here we report the initial characterization of the mutant phenotype. The cpnA ؊ cells exhibited normal growth rates and a slight cytokinesis defect. When placed in starvation conditions, cpnA ؊ cells appeared to aggregate into mounds and form fingers with normal timing; however, they were delayed or arrested in the finger stage. When placed in water, cpnA ؊ cells formed unusually large contractile vacuoles, indicating a defect in contractile vacuole function, while endocytosis and phagocytosis rates for the cpnA ؊ cells were similar to those seen for wild-type cells. These studies indicate that CpnA plays a role in cytokinesis and contractile vacuole function and is required for normal development, specifically in the later stages prior to culmination. We also used real-time reverse transcription-PCR to determine the expression patterns of all six copine genes during development.

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.

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.Protein phosphatase 1 (PP-1, 1 EC 3.13.16) dephosphorylates serine and threonine residues on proteins that control a diverse range of cellular processes from metabolism and muscle contraction to the cell cycle and gene expression (1). The involvement of PP-1 in all of these events raises the question as to how all of these processes can be regulated independently of one another? This question is even more apparent when one considers that the catalytic subunit of PP-1 (PP-1C) is expressed at micromolar concentrations in cells and shares 49% sequence homology within its catalytic core with at least 3 other highly expressed phosphatases, PP-2A, PP-2B, and PP-5 (2-5). The key to this paradox is the finding that the functions of PP-1 are closely linked to its subcellular localization with regulatory targeting subunits that confer substrate specificity (1). At the time of writing, 10 regulatory subunits have been sequenced at the protein and DNA level in mammalian tissues, and at least 10 others identified in Saccharomyces cerevisiae (6). Five of the mammalian subunits potently inhibit the enzyme's activity toward all substrates, whereas the others target the activity of PP-1C toward myosin, glycogen synthase, p53, or the nucleus (6). The small number of PP-1 regulatory proteins discovered thus far is unlikely to account for all the intracellular actions of the phosphatase, suggesting that many others must exist.One of the mechanisms by which PP-1C recognizes its regulatory subunits was recently characterized (6). Amino acid sequence alignments of nine of the mammalian regulatory subunits identifie...

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SummaryRecently, it has become clear that the actin cytoskeleton is involved in clathrin-mediated endocytosis. During clathrin-mediated endocytosis, clathrin triskelions and adaptor proteins assemble into lattices, forming clathrin-coated pits. These coated pits invaginate and detach from the membrane, a process that requires dynamic actin polymerization. We found an unexpected role for the clathrin adaptor epsin in regulating actin dynamics during this late stage of coated vesicle formation. In Dictyostelium cells, epsin is required for both the membrane recruitment and phosphorylation of the actin-and clathrin-binding protein Hip1r. Epsin-null and Hip1r-null cells exhibit deficiencies in the timing and organization of actin filaments at clathrin-coated pits. Consequently, clathrin structures persist on the membranes of epsin and Hip1r mutants and the internalization of clathrin structures is delayed. We conclude that epsin works with Hip1r to regulate actin dynamics by controlling the spatial and temporal coupling of actin filaments to clathrin-coated pits. Specific residues in the ENTH domain of epsin that are required for the membrane recruitment and phosphorylation of Hip1r are also required for normal actin and clathrin dynamics at the plasma membrane. We propose that epsin promotes the membrane recruitment and phosphorylation of Hip1r, which in turn regulates actin polymerization at clathrin-coated pits.