Cellular distribution of sarcoplasmic calcium-binding proteins by immunofluorescence

Summary Specific antibodies against carp paravalbumin, crayfish calcium binding protein and crayfish arginine kinase were used for indirect immunofluorescence localization of the respective proteins. Simultaneous staining of the same muscle sections with human serum containing anti-actin autoantibodies served as a probe to identify the isotropic band.Parvalbumin appears to be evenly distributed in carp white muscle. The crayfish calcium binding protein however shows a distinct localization, in the isotropic band, coincident with the actin staining. Arginine kinase, which has the same molecular weight and is extractible in the same way as the calcium binding protein, does not show this distinct localization, but is evenly present in crayfish tail muscle, similarly to parvalbumin.The possible meaning of the different distribution of the two calcium binding proteins is discussed.A preliminary report on this work was presented at the meeting of the Union of Swiss Societies for experimental Biology, Lausanne, 1975. This work was supported by the Swiss National Science Foundation, grants No. 3.725.72 and 3.0330.73     

Primary structure of three minor isoforms of amphioxus sarcoplasmic calcium-binding proteins.

Previously we reported the amino acid sequences of 4 well-defined sacroplasmic, high-affinity Ca(2+)-binding proteins in the protochordate amphioxus, Branchiostoma lanceolatum [1]. Here we report on the complete amino acid sequence determination of 3 additional minor isoforms. The seven isoforms differ from each other in 9 positions of a contiguous 17-residue-long segment (positions 20-36) and can be classified in a alpha (ASCP I, III and IV) and a beta lineage (ASCP II, V, VI and VII).     

Localization of sarcoplasmic reticulum proteins in rat skeletal muscle by immunofluorescence

Ca++-Mg++-dependent ATPase and calsequestrin, the major intrinsic and extrinsic proteins, respectively, of the sarcoplasmic reticulum, were localized in cryostat sections of adult rat skeletal muscle by immunofluorescent staining and phase-contrast microscopy. Relatively high concentrations of both the ATPase and calsequestrin were found in fast-twitch myofibers while a very low concentration of the ATPase and a moderate concentration of calsequestrin were found in slow-twitch myofibers. These findings are consistent with previous biochemical studies of the isolated sarcoplasmic reticulum of slow-twitch and fast-twitch mammalian muscles. The distribution of the ATPase in muscle fibers is distinctly different from that of calsequestrin. While calsequestrin is present only near the interface between the I- and A-band regions of the sarcomere, the ATPase is found throughout the I-band region as well as in the center of the A-band region. In comparing these results with in situ ultrastructural studies of the distribution of sarcoplasmic reticulum in fast-twitch muscle, it appears that the ATPase is rather uniformly distributed throughout the sarcoplasmic reticulum while calsequestrin is almost exclusively confined to those regions of the membrane system which correspond to terminal cisternae. Fluorescent staining with these antisera was not observed in vascular smooth muscle cells present in the cryostat sections of the mammalian skeletal muscle used in this study.     

Cellular distribution of p68, a new calcium-binding protein from lymphocytes.

A Ca2+-binding protein of mol. wt. 68 000 ( p68 ) is a major component of a Nonidet P-40 insoluble fraction of human and pig lymphocyte plasma membrane. An affinity-purified rabbit antibody has been produced against p68 and used to study its cellular distribution. The antibody stained fixed and permeabilised human B lymphoblastoid cells, peripheral blood lymphocytes and sections of human tonsil. Whole cells, however, were not stained, indicating that the protein was not represented at the cell surface. This assignment was consistent with the detection of p68 in immunoprecipitates from biosynthetically- but not surface-labelled cells. It is concluded that p68 is located on the cytoplasmic face of the plasma membrane. Subcellular fractionation experiments confirmed that p68 was largely membrane-bound in lymphocytes, although a small soluble fraction (approximately 10% of the total) was detected. Sub-fractionation of lymphocyte membranes revealed that p68 was associated not only with the plasma membrane but also with other endomembrane systems. As judged by immunoprecipitation, p68 was present in a variety of cultured cell lines of both lymphoid and non-lymphoid origin. p68 demonstrated a diffuse distribution in fixed and permeabilised fibroblasts which did not correspond to the distribution of either microfilaments or intermediate filaments. However, in detergent-extracted cells the protein was localised in a lamina-like network. A similar immunofluorescent staining pattern has recently been observed for spectrin-related proteins in the detergent-resistant cytoskeleton of fibroblasts. It is suggested that p68 is part of a sub-membranous cytoskeletal complex not only in lymphocytes but also in other cell types.     

Modulation of membrane fusion by calcium-binding proteins.

The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating fusion.     

Internal calcium-binding proteins

Ca(2+)-ions play a key role in the regulation of many cellular processes and impairment of calcium homeostasis has been implicated in several diseases. Intracellularly the Ca(2+)-signal is transmitted by two families of proteins, the 'EF-hand'- and the Ca(2+)-dependent and phospholipid binding proteins. Their protein and gene structures as well as possible functional roles are summarized.     

EF-hand calcium-binding proteins

The EF-hand motif is the most common calcium-binding motif found in proteins. Several high-resolution structures containing different metal ions bound to EF-hand sites have given new insight into the modulation of their binding affinities. Recently determined structures of members of several newly identified protein families that contain the EF-hand motif in some of their domains, as well as of their complexes with target molecules, are throwing light on the surprising variety of functions that can be served by this simple and ingenious structural motif.     

Assembly of the sarcoplasmic reticulum proteins in differentiating rat skeletal muscle cell cultures: localization by immunofluorescence of sarcoplasmic reticulum proteins in differentiating rat skeletal muscle cell cultures

Immunofluorescent staining techniques were used to study the distribution of the Ca(2) + Mg(2+)-dependent ATPase and calsequestrin in primary cultures of differentiating rat skeletal muscle cells, grown for different periods of time under various culture conditions. In mononucleated myoblasts calsequestrin was detected after 45 h in culture whereas the ATPase was not detected until 60 h. After cell fusion began, both proteins could be identified in all multinucleated cells. Myoblasts grown for longer than 60 h in low Ca(2+) medium contained calsequestrin and the ATPase, even though they were unable to fuse. These studies at the cellular level confirm biochemical findings on the biosynthesis of calsequestrin and the ATPase. Immunofluorescent staining of myoblasts showed that calsequestrin first appears in a well-defined region of the cell near one end of the nucleus. At later times, the staining occupied progressively larger regions adjacent to the nucleus and took on a fibrous appearance. This suggests that calsequestrin first accumulates in the Golgi region and then gradually spreads throughout the cell. In contrast, the ATPase appeared to be concentrated in many small patches or foci throughout the cytoplasm and was never confined to one particular region, although some parts of the cell often stained more intensely than others. In multinucleated cells, alternating dark and fluorescent strands parallel to the longitudinal axis of the cells were evident.     

Calcium-dependent proteolysis of calcium-binding proteins

In several myopathic disorders, the internal muscle cell calcium concentration increases significantly as compared to normal muscle cells. We report that in the presence of elevated calcium levels, the calcium-binding proteins troponin C and calmodulin are protected from digestion by the chymotrypsin-like serine proteinase that co-purifies with isolated myofibrils. Degradation of the 67k calcimedin in the presence of calcium shows altered major cleavage fragments while degradation of myosin is unaffected by the presence of calcium. A role for this serine proteinase in muscle-wasting diseases is suggested.     

Characterization of vacuolar calcium-binding proteins

The vacuole plays a major structural and biochemical role in the higher plant cell. Among the most studied properties of the vacuole have been transport activities. One important aspect of vacuolar function is its participation in the regulation of cytosolic calcium levels. To identify the molecular entities involved in calcium regulation, a study of vacuole-associated, calcium-binding proteins (CaBs) was initiated. A competition assay was used, and it was observed that the majority of the total cellular membrane-associated, calcium-binding activity resided in low-density fractions enriched in vacuole membranes. Much of that calcium-binding activity was inactivated by a 0.5 m KI wash, and of the remaining activity, 77% was estimated to be peripherally associated with vacuolar membranes, whereas 23% was integrally associated with the vacuolar membrane. Calcium-ligand blots were used, and four major CaBs, with apparent molecular masses of 64, 58, 55, and 42 kD, were detected in purified vacuole membrane fractions. Two of these, the 58- and the 55-kD polypeptide, also appear to be present in significant amounts in endoplasmic reticulum-enriched fractions. However, the 64- and the 42-kD polypeptide are found primarily in vacuolar fractions. It is interesting that expression of the 42-kD polypeptide appears to be restricted to the heavily vacuolated cortical tissues (i.e. it is not found in vascular tissues). The localization of CaBs in the vacuole is consistent with the presence of calcium uptake (H⁺/Ca²⁺ antiport) and release mechanisms (inositol trisphosphate sensitive) on vacuolar membranes. These vacuole-associated CaBs, which may play a role in calcium buffering, together with the calcium transport systems, could mediate the vacuolar component of cellular calcium homeostasis.     

Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins.

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases identified as being critical for the regulation of cell division and cell morphology. The regulatory mechanisms that control Ndr activity have not been characterized previously. In this paper, we present evidence that Ndr is regulated by EF-hand calcium-binding proteins of the S100 family, in response to changes in the intracellular calcium concentration. In vitro, S100B binds directly to and activates Ndr in a Ca2+-dependent manner. Moreover, Ndr is recovered from cell lysates in anti-S100B immunoprecipitates. The region of Ndr responsible for interaction with Ca2+/S100B is a basic/hydrophobic motif within the N-terminal regulatory domain of Ndr, and activation of Ndr by Ca2+/S100B is inhibited by a synthetic peptide derived from this region. In cultured cells, Ndr is rapidly activated following treatment with Ca2+ ionophore, and this activation is dependent upon the identified Ca2+/S100B-binding domain. Finally, Ndr activity is inhibited by W-7 in melanoma cells overexpressing S100B, but is unaffected by W-7 in melanoma cells that lack S100B. These results suggest that Ndr is regulated at least in part by changes in the intracellular calcium concentration, through binding of S100 proteins to its N-terminal regulatory domain.     

The annexin family of calcium-binding proteins. Review article

The annexins are a family of calcium-binding proteins. Data from protein and cDNA sequencing have shown that at least five distinct but closely related mammalian annexins exist each of which possesses four or eight homologous internal repeats which may be calcium-and phospholipid-binding domains. The proteins are present within a wide range of tissues and cell types, with each cell type having all or a subset of the proteins. The proteins are localised on the inner surface of the plasma membrane associated with the cytoskeleton and in some cases also with intracellular structures. Some members of the family are major substrates for tyrosine and serine kinases. The precise functions of the proteins are unknown but they are likely to play important roles in cellular regulation. Previously suggested functions are inhibition of phospholipase A2, membrane-cytoskeletal linkage and control of membrane fusion events in exocytosis. It is also suggested that they may be involved in the regulation of cell surface receptors.     

Three-dimensional structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor

The three-dimensional structure of a sarcoplasmic Ca2(+)-binding protein from the sandworm Nereis diversicolor has been determined at 3.0 A resolution using multiple isomorphous replacement techniques. The NH2-terminal half of the molecule contains one variant Ca2(+)-binding domain with a novel helix-loop-helix conformation and one Ca2(+)-binding domain that is no longer functional because of amino acid changes. The overall conformation of this pair of domains is different from any previously described Ca2(+)-binding protein. The COOH-terminal half of the protein contains two Ca2(+)-binding domains with the usual helix-loop-helix configuration and is similar to calmodulin and troponin C. Unlike calmodulin or troponin C, there is no exposed alpha-helix connecting the two halves of the molecule, so the overall structure is much more compact.     

PINOID-mediated signaling involves calcium-binding proteins

The plant hormone auxin is a central regulator of plant development. In Arabidopsis, the PINOID (PID) protein serine/threonine kinase is a key component in the signaling of this phytohormone. To further investigate the biological function of PID, we performed a screen for PID-interacting proteins using the yeast two-hybrid system. Here, we show that PID interacts with two calcium-binding proteins: TOUCH3 (TCH3), a calmodulin-related protein, and PID-BINDING PROTEIN 1 (PBP1), a previously uncharacterized protein containing putative EF-hand calcium-binding motifs. The interaction between PID and the calcium-binding proteins is significant because it is calcium dependent and requires an intact PID protein. Furthermore, the expression of all three genes (PID, TCH3, and PBP1) is up-regulated by auxin. TCH3 and PBP1 are not targets for phosphorylation by PID, suggesting that these proteins act upstream of PID. PBP1 was found to stimulate the autophosphorylation activity of PID, and calcium influx and calmodulin inhibitors where found to enhance the activity of PID in vivo. Our results indicate that TCH3 and PBP1 interact with the PID protein kinase and regulate the activity of this protein in response to changes in calcium levels. This work provides the first molecular evidence for the involvement of calcium in auxin-regulated plant development.     

Lead-binding properties of intestinal calcium-binding proteins

The bovine and chick vitamin D-induced intestinal calcium-binding proteins (CaBP) bind lead. Bovine CaBP binds 2 atoms of lead/molecule, and chick CaBP binds 4 atoms of lead per molecule and these values are identical to those for calcium binding. 45Calcium-displacement studies indicate significantly higher affinities for lead than for calcium for both proteins. All evidence indicates that lead is bound to the 4 high affinity calcium-binding sites on chick CaBP and to the corresponding 2 high affinity sites on bovine CaBP, and that binding of lead to sulfhydryl groups is, relatively, not significant. Calmodulin, troponin C, and oncomodulin also bind lead with high affinities and in preference to calcium, indicating that lead binding is a general property of proteins belonging to the troponin C superfamily of calcium-binding proteins.