Immunolocalization of a novel, cytoskeleton-associated polypeptide of Mr 230,000 daltons (p230)

Antibodies were raised against a cytoskeleton-associated, nonphosphorylated, 230,000-dalton bovine lens polypeptide (designated p230), and rendered monospecific by using a novel immunoaffinity technique. In immunofluorescence and electron microscopy of cultured fibroblasts, as well as of various other cells (endothelial, epithelial, lenticular, monocytes, neuroblastoma cells) and tissues (human kidney and liver), p230 was localized as a distinct subplasmalemmal layer in the peripheral cytoplasm of the cells. It constituted less than 0.3% of the total cellular protein in cultured fibroblasts and was not extractable with Triton X-100. In detergent-extracted cytoskeletal preparations of cultured fibroblasts, p230 remained as an elaborate peripheral network that showed a distribution distinctly different from that of the major cytoskeletal structures, stress fibers, cortical myosin, vinculin, and intermediate filaments (IF). The distribution was not dependent on the presence of intact stress fibers or microtubules, as shown by double-fluorescence microscopy of cells exposed to cytochalasin B or cultured in the presence of monensin and of cold-treated cells. Upon demecolcine-induced reorganization of intermediate filaments, however, the localization of p230 was rapidly altered to a dense plaque underneath the perinuclear aggregate of intermediate filaments. On the other hand, p230 seemed to colocalize with the detergent-resistant cell surface lamina, visualized in fluorescence microscopy with fluorochrome-coupled wheat germ agglutinin-lectin. The results suggest that p230 is part of a cell surface- and cytoskeleton-associated subplasmalemmal structure that may play an important role in cell surface-cytoskeleton interaction in various cells both in vitro and in vivo.     

Localization of the 230-kilodalton bullous pemphigoid antigen in cultured keratinocytes: formation of a prehemidesmosome.

The hemidesmosome is the major attachment structure of the epidermal basal cell visible ultrastructurally in skin. The importance of its components to cultured cell attachment to substratum is not understood, however. A component of the hemidesmosome, the 230-kDa bullous pemphigoid antigen (p230), has been shown to be present in an insoluble or particulate fraction of cultured cells. In order to more fully characterize its potential importance for cell-matrix adhesion in cultured keratinocytes, specific antibodies were raised to the C-terminal region of p230 expressed as a bacterial fusion protein. Such antibodies recognize the hemidesmosome of epidermis, binding on the cytoplasmic region of its plaque. In addition, keratinocytes cultured in a 0.15 mM Ca(2+)-defined medium contain a detergent-resistant pool of p230 which appears to lie in the same focal plane as the culture substrate and has a patchy or irregular distribution by indirect immunofluorescence. Treatment of cultured cells at 4 degrees C with trypsin or pronase sufficient to release keratinocytes from the culture dish does not affect the electrophoretic migration of p230 on SDS-gels, suggesting that p230 is not exposed to the extracellular space. In cells cultured in 0.15 mM Ca2+, 230-kDa BP antigen is localized to discrete clusters resting near the basal plasma membrane of the cell by immunogold staining following brief detergent treatment and fixation. These clusters are approximately 0.1 micron in diameter, which is similar in size to the in vivo hemidesmosome. Fully formed electron dense hemidesmosomal plaques are not observed under the same culture conditions, however. It appears that these clusters are early precursors of the hemidesmosome.     

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.     

Quantitative immunofluorescence, anti-ras p21 antibody specificity, and cellular oncoprotein levels

A general approach to investigating specificity and saturation of antibodies by quantitative immunofluorescence is applied to monoclonal antibodies generated against p21 or ras oligopeptides to quantify ras p21 oncoprotein in cultured cells. Ras 10, a panreactive mouse monoclonal antibody, appears to be a superior probe for detection of p21 in cell extracts or fixed cells because it binds a 21 kD protein on SDS-PAGE/western blots and labels the cytoplasmic membrane in a saturable and competitive manner. RAP-5, a widely used mouse monoclonal antibody generated against an oligopeptide of ras p21, does not recognize p21 in denaturing immunoblots or in immunofluorescence of cultured cells.     

Interaction between p230 and MACF1 is associated with transport of a glycosyl phosphatidyl inositol-anchored protein from the Golgi to the cell periphery

The molecular basis by which proteins are transported along cytoskeletal tracts from the trans-Golgi network (TGN) to the cell periphery remains poorly understood. Previously, using human autoimmune sera, we identified and characterized a TGN protein, p230/Golgin-245, an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, that is anchored to TGN membranes and TGN-derived vesicles by its carboxyl-terminal GRIP domain. To identify molecules that interact with the flexible amino-terminal end of p230, we used this domain as bait to screen a human brain cDNA library in a yeast two-hybrid assay. We found that this domain interacts with the carboxyl-terminal domain of MACF1, a protein that cross-links microtubules to the actin cytoskeleton. The interaction was confirmed by co-immunoprecipitation, an in vitro binding assay, double immunofluorescence images demonstrating overlapped localization in HeLa cells, and co-localization of FLAG-tagged constructs containing the interacting domains of these two proteins with their endogenous partners. Expression in HeLa cells of FLAG-tagged constructs containing the interacting domains of p230 and MACF1 disrupted transport of the glycosyl phosphatidyl inositol-anchored marker protein conjugated with yellow fluorescent protein (YFP-SP-GPI), while trafficking of the transmembrane marker protein, vesicular stomatitis virus glycoprotein conjugated with YFP (VSVG3-GL-YFP), was unaffected. Our results suggest that p230, through its interaction with MACF1, provides the molecular link for transport of GPI-anchored proteins along the microtubule and actin cytoskeleton from the TGN to the cell periphery.     

The submembranous location of p11 and its interaction with the p36 substrate of pp60 src kinase in situ

p36, a major cytoplasmic substrate of pp60 src kinase, is present beneath the plasma membrane. It can be isolated either as a monomer or as a heterotetramer (protein I) containing two copies each of p36 and a unique p11 polypeptide. To compare the expression rules of p36 and p11 as well as their cellular distributions, monoclonal antibodies to the two porcine proteins were isolated. In tissue culture cells p11-specific antibodies decorated the same submembranous compartment previously seen with antibodies to p36 and fodrin or spectrin and followed the p36 images under all fixation/extraction conditions tested. Immunofluorescence microscopy on tissue sections showed coincident expression patterns of both proteins confirming and extending previous results with p36 antibodies. Antibodies with limited cross-species reaction have been used to trace the fate of porcine p11 and p36 injected into cultured cells. Both proteins are incorporated in the submembranous compartment, where they remain in Triton cytoskeletons prepared in the presence but not in the absence of Ca2+. The incorporation of p36 in vivo conforms with its Ca2+-dependent binding to actin, fodrin, and certain phospholipids in vitro. In contrast, the incorporation of p11 seems to depend on an in situ interaction with p36 or an exchange with endogenous p11 present on p36. The combined results indicate a strong coupling of p11 and p36 in cellular compartmentalization and tissue differentiation.     

p115 Interacts with the GLUT4 vesicle protein, IRAP, and plays a critical role in insulin-stimulated GLUT4 translocation

Insulin-regulated aminopeptidase (IRAP) is an abundant cargo protein of Glut4 storage vesicles (GSVs) that traffics to and from the plasma membrane in response to insulin. We used the amino terminus cytoplasmic domain of IRAP, residues 1-109, as an affinity reagent to identify cytosolic proteins that might be involved in GSV trafficking. In this way, we identified p115, a peripheral membrane protein known to be involved in membrane trafficking. In murine adipocytes, we determined that p115 was localized to the perinuclear region by immunofluorescence and throughout the cell by fractionation. By immunofluorescence, p115 partially colocalizes with GLUT4 and IRAP in the perinuclear region of cultured fat cells. The amino terminus of p115 binds to IRAP and overexpression of a N-terminal construct results in its colocalization with GLUT4 throughout the cell. Insulin-stimulated GLUT4 translocation is completely inhibited under these conditions. Overexpression of p115 C-terminus has no significant effect on GLUT4 distribution and translocation. Finally, expression of the p115 N-terminus construct has no effect on the distribution and trafficking of GLUT1. These data suggest that p115 has an important and specific role in insulin-stimulated Glut4 translocation, probably by way of tethering insulin-sensitive Glut4 vesicles at an as yet unknown intracellular site.     

Identity of p36K phosphorylated upon Rous sarcoma virus transformation with a protein purified from brush borders; calcium-dependent binding to non-erythroid spectrin and F-actin.

Membrane vesicles derived from the apical side of procine intestinal epithelial cells retain, after demembranation in the presence of calcium, two major proteins (I, II) which are released by the addition of calcium chelators. We have purified and characterized these two calcium-binding proteins. Protein I has a mol. wt. of 85 000 and contains two copies of a 36-K subunit and an additional 10-K subunit. It binds in a calcium-dependent manner to F-actin as well as to non-erythroid spectrin. Immunofluorescence microscopy reveals protein I-related antigens in the terminal web of the intestinal cell and in a submembraneous cortical layer in various tissue culture cells. Biochemical and immunological results document that the 36-K subunit of protein I is identical with the cellular p36K recognized as a major substrate for tyrosine phosphorylation by the sarc gene kinase in Rous sarcoma virus-transformed cells. The biochemical properties of protein I agree with its location seen in immunofluorescence microscopy and cell fractionation and suggest that the actin-spectrin network in the cortical layer may be affected by virus transformation.     

Raft partitioning of the yeast uracil permease during trafficking along the endocytic pathway

Lipid rafts, formed by the lateral association of sphingolipids and cholesterol in the external membrane leaflet, have been implicated in membrane traffic and cell signaling in mammalian cells. Yeast plasma membranes were also recently shown to contain lipid raft microdomains consisting of sphingolipids and ergosterol, and containing several plasma membrane proteins, including Gas1p, a GPI-anchored protein, and the [H⁺] ATPase Pma1p. In this study, we investigated whether lipid rafts were involved in the intracellular trafficking of a yeast transporter, uracil permease, which undergoes ubiquitin-dependent endocytosis. Regardless of its ubiquitination status, uracil permease was found to be associated with rafts in the plasma membrane. The expression of Fur4p in lcb1–100 cells, deficient in the first enzyme of sphingolipid synthesis, impaired the association of Fur4p with detergent-resistant fractions. When targeted to endocytic compartments, uracil permease appeared to be progressively transferred to detergent-soluble fractions, suggesting that the lipid environment might change between plasma membrane and endosomes. Consistent with this hypothesis, the wild-type form of the v-SNARE Snc1p, which is known to cycle between the plasma membrane and endosomal compartments, was recovered in both detergent-resistant and detergent-soluble fractions. In contrast, a variant Snc1p that accumulates at the plasma membrane was recovered exclusively in detergent-resistant fractions .     

Reorganization of plasma membrane-associated 36 000 dalton protein upon drug-induced redistribution of cytokeratin

Epithelial PtK2 cells were used as a model to study the possible redistribution of the major tyrosine protein kinase substrate, p36, upon drug-induced reorganization of the cytokeratin network. Cells were grown on glass coverslips and exposed to cytochalasin D (CD), colcemid or a combination of the two. The cytokeratin type intermediate filaments of the cells were redistributed by treatment of the cells with colchicine and CD. Simultaneous changes in cytokeratin and in p36 antigen were observed by double-label immunofluorescence. In control PtK2 cells, p36 was distributed characteristically at the cortical cytoplasm. Neither colchicine nor CD alone was able to cause a major reorganization of cytokeratin or p36. Their combined effect resulted in formation of blebs containing abundant p36 at the cell surface and at cell-cell junctions. Actin, on the other hand, was reorganized to similar configurations by CD alone. These observations show for the first time a drug-induced redistribution of p36. The results suggest a relationship between membrane-associated p36 and the cytoskeletal fibres that terminate at the plasma membrane.     

Co-precipitation of intestinal p36 with a 73-K protein and a high molecular weight factor

p36 is a major substrate of tyrosine kinases that co-localizes with spectrin in nonerythroid cells. Recent studies by Gerke & Weber [14] have shown that p36 can be isolated from intestine by selective extraction with the Ca2+-chelating agent EGTA. We now show that p36 can be re-precipitated by adding free Ca2+ to 1 mM with the co-precipitation of a high molecular weight (MW) factor and a polypeptide of 73K. The 73K protein was purified to apparent homogeneity, rabbit antibodies were raised to it and used in Western blots and immunofluorescence microscopy. The 73K protein is found in a wide range of tissues and is particularly concentrated in fibroblasts, where its distribution partially overlaps that of non-erythroid spectrin.     

Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells

Annexin II is a Ca(2+)-dependent membrane-binding protein present in a wide variety of cells and tissues. Within cells, annexin II is found either as a 36-kD monomer (p36) or as a heterotetrameric complex (p90) coupled with the S-100-related protein, p11. Annexin II has been suggested to be involved in exocytosis as it can restore the secretory responsiveness of permeabilized chromaffin cells. By quantitative confocal immunofluorescence, immunoreplica analysis and immunoprecipitation, we show here the translocation of p36 from the cytosol to a subplasmalemmal Triton X-100 insoluble fraction in chromaffin cells following nicotinic stimulation. A synthetic peptide corresponding to the NH2-terminal domain of p36 which contains the phosphorylation sites was microinjected into individual chromaffin cells and catecholamine secretion was monitored by amperometry. This peptide blocked completely the nicotine-induced recruitment of p36 to the cell periphery and strongly inhibited exocytosis evoked by either nicotine or high K+. The light chain of annexin II, p11, was selectively expressed by adrenergic chromaffin cells, and was only present in the subplasmalemmal Triton X-100 insoluble protein fraction of both resting and stimulated cells. p11 can modify the Ca(2+)- and/or the phospholipid-binding properties of p36. We found that loss Ca2+ was required to stimulate the translocation of p36 and to trigger exocytosis in adrenergic chromaffin cells. Our findings suggest that the translocation of p36 to the subplasmalemmal region is an essential event in regulated exocytosis and support the idea that the presence of p11 in adrenergic cells may confer a higher Ca2+ affinity to the exocytotic pathway in these cells.     

PrPC is sorted to the basolateral membrane of epithelial cells independently of its association with rafts

PrP(C) is a glycosylphosphatidylinositol-anchored protein expressed in neurons as well as in the cells of several peripheral tissues. Although the normal function of PrP(C) remains unknown, a conformational isoform called PrP(Sc) (scrapie) has been proposed to be the infectious agent of transmissible spongiform encephalopathies in animals and humans. Where and how the PrP(C) to PrP(Sc) conversion occurs in the cells is not yet known. Therefore, dissecting the intracellular trafficking of the wild-type prion protein, as well as of the scrapie isoform, can be of major relevance to the pathogenesis of the diseases. In this report we have analyzed the exocytic pathway of transfected mouse PrP(C) in thyroid and kidney polarized epithelial cells. In contrast to the majority of glycosylphosphatidylinositol-anchored proteins, we found that PrP(C) is localized mainly on the basolateral domain of the plasma membrane of both cell lines. This is reminiscent of the predominant somatodendritic localization found in neurons. However, similarly to apical glycosylphosphatidylinositol-proteins, PrP(C) associates with detergent-resistant microdomains, which have been suggested to have a role in apical sorting of glycosylphosphatidylinositol-proteins, as well as in the conversion process of PrP(C) to PrP(Sc). In order to discriminate whether detergent-resistant microdomains have a direct role in PrP(Sc) conversion, or whether they are involved in the transport of the protein to the site of its conversion, we have examined the effect of disruption of detergent-resistant microdomain association on PrP(C) intracellular traffic. Consistent with the unusual basolateral localization of this glycosylphosphatidylinositol-linked protein, our data exclude a classical role for detergent-resistant microdomains in the post-trans-Golgi network sorting and transport of PrP(C) to the plasma membrane.     

Subcellular location of an abundant substrate (p36) for tyrosine-specific protein kinases.

A 36,000-dalton cellular protein (p36) has been identified previously as an abundant substrate for phosphorylation by tyrosine-specific protein kinases. Since several of the responsible kinases are associated with the plasma membrane, we explored the subcellular distribution of p36. Biochemical fractionations located p36 on the plasma membrane of both normal and retrovirus-transformed cells. Approximately half of the p36 was bound to the membrane with the affinity of a peripheral membrane protein; the remainder was even more tightly bound. The distribution of p36 among subcellular fractions and its affinity for the plasma membrane were not affected by tyrosine phosphorylation. We determined that p36 is synthesized in the soluble compartment of the cell and then moves rapidly to the membranous compartment. Immunofluorescence microscopy with antibodies directed against p36 revealed two distinct distributions of the antigen: (i) a sharply demarcated crenelated pattern within or immediately beneath the plasma membrane, which we presume to be a correlary of the distribution of p36 in biochemical fractionations; and (ii) diffuse staining in a cytoplasmic location that could not be attributed to a specific feature of cytoarchitecture and could not be easily reconciled with the results of biochemical fractionations. Efforts to detect the secretion of p36 were unsuccessful. No evidence was obtained for exposure of p36 on the cell surface, and no changes in localization were observed as a consequence of neoplastic transformation. During the course of this study, we had the opportunity to pursue a previous report that p36 is a component of the enzyme malate dehydrogenase (Rubsamen et al., Proc. Natl. Acad. Sci. U.S.A. 79:228-232, 1982). We were unable to substantiate this claim. We conclude that at least a substantial fraction of p36 is located on the cytoplasmic aspect of the plasma membrane, where it could be well situated to serve as a substrate for several identified tyrosine-specific kinases. But the function of p36 and its role, if any, in neoplastic transformation of cells by retroviruses possessing tyrosine-specific kinases remain enigmatic.     

The Dynamin-related GTPase,Dnm1p,Controls Mitochondrial Morphology in Yeast

The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.     

Glycosylphosphatidylinositol-anchored proteins are required for the transport of detergent-resistant microdomain-associated membrane proteins Tat2p and Fur4p

In eukaryotic cells many cell surface proteins are attached to the membrane via the glycosylphosphatidylinositol (GPI) moiety. In yeast, GPI also plays important roles in the production of mannoprotein in the cell wall. We previously isolated gwt1 mutants and found that GWT1 is required for inositol acylation in the GPI biosynthetic pathway. In this study we isolated a new gwt1 mutant allele, gwt1-10, that shows not only high temperature sensitivity but also low temperature sensitivity. The gwt1-10 cells show impaired acyltransferase activity and attachment of GPI to proteins even at the permissive temperature. We identified TAT2, which encodes a high affinity tryptophan permease, as a multicopy suppressor of cold sensitivity in gwt1-10 cells. The gwt1-10 cells were also defective in the import of tryptophan, and a lack of tryptophan caused low temperature sensitivity. Microscopic observation revealed that Tat2p is not transported to the plasma membrane but is retained in the endoplasmic reticulum in gwt1-10 cells grown under tryptophan-poor conditions. We found that Tat2p was not associated with detergent-resistant membranes (DRMs), which are required for the recruitment of Tat2p to the plasma membrane. A similar result was obtained for Fur4p, a uracil permease localized in the DRMs of the plasma membrane. These results indicate that GPI-anchored proteins are required for the recruitment of membrane proteins Tat2p and Fur4p to the plasma membrane via DRMs, suggesting that some membrane proteins are redistributed in the cell in response to environmental and nutritional conditions due to an association with DRMs that is dependent on GPI-anchored proteins.     

Characterization of A 54-kD protein of the inner nuclear membrane: evidence for cell cycle-dependent interaction with the nuclear lamina

Using a mAb (R-7), we have characterized a 54-kD protein of the chicken nuclear envelope. Based on its biochemical properties and subnuclear distribution p54 is likely to be an integral membrane component specific to the inner nuclear membrane. Fractionation experiments indicate that p54 interacts, directly or indirectly, with the nuclear lamina, and analysis of p54 in cultured cells suggests that this interaction is controlled by cell cycle-dependent posttranslational modification, most likely phosphorylation. Modification of p54 results in a slightly reduced electrophoretic mobility, and it converts the protein from a detergent-resistant to a detergent-extractable form. Detergent solubilization of p54 can be induced in vivo by treating isolated nuclei or nuclear envelopes with highly purified cdc2 kinase, one of the most prominent kinases active in mitotic cells. These results suggest that mitotic phosphorylation of p54 might contribute to control nuclear envelope dynamics during mitosis in vivo.     

Characterization of a multidomain adaptor protein, p140Cap, as part of a pre-synaptic complex

p140Cap (Cas-associated protein) is an adaptor protein considered to play pivotal roles in cell adhesion, growth and Src tyrosine kinase-related signaling in non-neuronal cells. It is also reported to interact with a pre-synaptic membrane protein, synaptosome-associated protein of 25 kDa, and may participate in neuronal secretion. However, properties and precise functions of p140Cap in neuronal cells are almost unknown. Here we show, using biochemical analyses, that p140Cap is expressed in rat brain in a developmental stage-dependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults. Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses of primary cultured rat hippocampal neurons. Electron microscopy further revealed localization at pre- and post-synapses. Screening of p140Cap-binding proteins identified a multidomain adaptor protein, vinexin, whose third Src-homology 3 domain interacts with the C-terminal Pro-rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a pre-synaptic protein, synaptophysin, interacts with p140Cap. These results suggest that p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling.     

Immunocytochemical localization of 67 KD Ca2+ binding protein (p67) in ventricular, skeletal, and smooth muscle cells.

By using immunocytochemical techniques, we examined the localization of a 67 kDa Ca2+ binding protein (p67) and calpactin I heavy chain (p36) in ventricular myocytes, skeletal myocytes, and intestinal smooth muscle cells. Immunofluorescence microscopy revealed that the p67 was expressed in all these muscle cells, whereas anti-p36 antibody stained cells in connective tissues but failed to stain these muscle cells. Immunogold electron microscopy was carried out to examine the subcellular localization of the p67 in muscle cells. The results showed that the p67 was exclusively confined to the plasma membrane of muscle cells and the presumptive transverse tubules of the striated myocytes. Immunoblot analysis with anti-p67 antibody showed that the p67 was indeed a constitutive protein of the sarcolemma isolated from rat hearts. These results indicate that the p67 is a sarcolemma-associated Ca2+ binding protein expressed in both striated myocytes and intestinal smooth muscle cells.     

GRIP domain-mediated targeting of two new coiled-coil proteins,GCC88 and GCC185, to subcompartments of the trans-Golgi network

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the trans-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of HeLa cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the trans-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.