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1.
The luminal SR protein CSQ2 contains phosphate on roughly half of the serines found in its C-terminus. The sequence around phosphorylation sites in CSQ2 suggest that the in vivo kinase is protein kinase CK2, even though this enzyme is thought to be present only in the cytoplasm and nucleus. To test whether CSQ2 kinase is CK2, we combined approaches that reduced CK2 activity and CSQ2 phosphorylation in intact cells. Tetrabromocinnamic acid, a specific inhibitor of CK2, inhibited both the CSQ2 kinase and CK2 in parallel across a range of concentrations. In intact primary adult rat cardiomyocytes and COS cells, 24 h of drug treatment reduced phosphorylation of overexpressed CSQ2 by 75%. Down-regulation of CK2α subunits in COS cells using siRNA, produced a 90% decrease in CK2α protein levels, and CK2-silenced COS cells exhibited a twofold reduction in CSQ2 kinase activity. Phosphorylation of CSQ2 overexpressed in CK2-silenced cells was also reduced by a factor of two. These data suggested that CSQ2 in intact cells is phosphorylated by CK2, a cytosolic kinase. When phosphorylation site mutants were analyzed in COS cells, the characteristic rough endoplasmic reticulum form of the CSQ2 glycan (GlcNAc2Man9,8) underwent phosphorylation site dependent processing such that CSQ2-nonPP (Ser to Ala mutant) and CSQ2-mimPP (Ser to Glu mutant) produced apparent lower and greater levels of ER retention, respectively. Taken together, these data suggest CK2 can phosphorylate CSQ2 co-translationally at biosynthetic sites in rough ER, a process that may result in changes in its subsequent trafficking through the secretory pathway.  相似文献   

2.
Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.  相似文献   

3.
Molecular mechanisms underlying Ca(2+) regulation by perinuclear endoplasmic/sarcoplasmic reticulum (ER/SR) cisternae in cardiomyocytes remain obscure. To investigate the mechanisms of changes in cardiac calsequestrin (CSQ2) trafficking on perinuclear Ca(2+) signaling, we manipulated the subcellular distribution of CSQ2 by overexpression of CSQ2-DsRed, which specifically accumulates in the perinuclear rough ER. Adult ventricular myocytes were infected with adenoviruses expressing CSQ2-DsRed, CSQ2-WT, or empty vector. We found that perinuclear enriched CSQ2-DsRed, but not normally distributed CSQ2-WT, enhanced nuclear Ca(2+) transients more potently than cytosolic Ca(2+) transients. Overexpression of CSQ2-DsRed produced more actively propagating Ca(2+) waves from perinuclear regions than did CSQ2-WT. Activities of the SR/ER Ca(2+)-ATPase and ryanodine receptor type 2, but not inositol 1,4,5-trisphosphate receptor type 2, were required for the generation of these perinuclear initiated Ca(2+) waves. In addition, CSQ2-DsRed was more potent than CSQ2-WT in inducing cellular hypertrophy in cultured neonatal cardiomyocytes. Our data demonstrate for the first time that CSQ2 retention in the rough ER/perinuclear region promotes perinuclear Ca(2+) signaling and predisposes to ryanodine receptor type 2-mediated Ca(2+) waves from CSQ2-enriched perinuclear compartments and myocyte hypotrophy. These findings provide new insights into the mechanism of CSQ2 in Ca(2+) homeostasis, suggesting that rough ER-localized Ca(2+) stores can operate independently in raising levels of cytosolic/nucleoplasmic Ca(2+) as a source of Ca(2+) for Ca(2+)-dependent signaling in health and disease.  相似文献   

4.
Skeletal muscle calsequestrin (skelCSQ) and cardiac calsequestrin (cardCSQ) are resident proteins of the ER/SR, but mechanisms by which CSQ is retained inside membrane lumens remain speculative. A structural model that predicts linear CSQ polymers has been developed that might explain CSQ concentration and localization inside junctional SR lumens, however little evidence exists for polymer formation in intact cells or for its effects on subcellular localization. We previously showed that cardCSQ is efficiently retained within the ER, but its retention is lost under conditions expected to disrupt its polymerization. In the present study, we found unexpectedly that skelCSQ shows no co-localization with cardCSQ in COS cells or in rat neonatal heart cells, but instead concentrates in a membrane compartment (ERGIC) that is just distal to that of cardCSQ. Consistent with this difference in immunofluorescent localization, the structures of CSQ (316Asn-linked) glycans showed two types of pre-Golgi processing. Despite the difference in subcellular distribution of individual wild-type forms of CSQ, however, pairs of different CSQ molecules (for example, different isoforms or different fluorescent fusion proteins) consistently co-localized, suggesting that separate forms of CSQ polymerize in different parts of the same secretory pathway, while different CSQ pairs localize together through heteropolymerization.  相似文献   

5.
Calsequestrin (CSQ) concentrates in junctional sarcoplasmic reticulum (SR) where it functions in regulation of Ca2+ release. When purified from heart tissue, cardiac CSQ contains phosphate on a cluster of C-terminal serine residues, but little is known about the cellular site of kinase action, and the identity of the kinase remains uncertain. To determine basic features of the phosphorylation, we examined the reaction in canine heart preparations. CSQ phosphorylation was observed in [32P]metabolically-labeled heart cells after adenoviral overexpression, and its constitutive phosphorylation was limited to a CK2-sensitive C-terminal serine cluster. The CSQ kinase was oriented intralumenally, as was CSQ, inside membrane vesicles, such that exposure to each required detergent permeabilization. Yet even after detergent permeabilization, CSQ was phosphorylated much less efficiently by protein kinase CK2 in cardiac microsomes than was purified CSQ. Reduced phosphorylation was strongly dependent upon protein concentration, and phosphorylation time courses revealed a phosphatase activity that occurred constitutively as phosphorylated substrate accumulates. Evidence of selective dephosphorylation of CSQ glycoforms in heart homogenates was also seen by mass spectrometry analysis. Molecules with greater mannose content, a feature of early secretory pathway compartments, were more highly phosphorylated, while greater dephosphorylation was apparent in more distal compartments. Taken together, the analyses of CSQ phosphorylation in heart suggest that a constitutive process of phosphate turnover occurs for cardiac CSQ perhaps associated with its intracellular transport.  相似文献   

6.
Calsequestrin (CSQ), the major low-affinity Ca(2+)-binding glycoprotein of striated muscle fibers, is concentrated to yield aggregates that occupy the lumen of the terminal cisternae of the sarcoplasmic reticulum (SR). When infected or transfected into L6 myoblast, the protein is also concentrated, however, in dense vacuoles apparently separate from the endoplasmic reticulum (ER). CSQ-rich cells appear otherwise normal; in particular, neither other proteins involved in Ca2+ homeostasis nor ER chaperones are increased. The CSQ dense vacuoles are shown herein to be specialized ER subdomains as demonstrated by 1) the endoglycosidase H sensitivity of their CSQ and 2) two markers, calreticulin and calnexin (but not others, protein disulfide isomerase and BiP), intermixed with the vacuole content. Their formation is shown to start with the aggregation of CSQ at discrete sites of the ER lumen. When cells were transfected with both CSQ and calreticulin, only the first gave rise to vacuoles; the second remained diffusely distributed within the ER lumen. The possibility that CSQ aggregation is an artifact of overexpression appears unlikely because 1) within dense vacuoles CSQ molecules are not disulfide cross-linked, 2) their turnover is relatively slow (t = 12 h), and 3) segregated CSQ is bound to large amounts of Ca2+. Transfection of a tagged CSQ into cells already overexpressing the protein revealed the continuous import of the newly synthesized protein into preassembled vacuoles. The tendency to aggregation appears, therefore, as a property contributing to the segregation of CSQ within the ER lumen and to its accumulation within specialized subdomains. The study of L6 cells expressing CSQ-rich vacuoles might thus ultimately help to unravel mechanisms by which the complexity of the sarcoplasmic reticulum is established in muscle fibers.  相似文献   

7.
Calsequestrin 2 (CSQ2) is generally regarded as the primary Ca2+-buffering molecule present inside the sarcoplasmic reticulum (SR) in cardiac cells, but findings from CSQ2 knockout experiments raise major questions about its role and necessity. This study determined the absolute amount of CSQ2 present in cardiac ventricular muscle to gauge its likely influence on SR free Ca2+ concentration ([Ca2+]) and maximal Ca2+ capacity. Ventricular tissue from hearts of freshly killed sheep was examined by SDS-PAGE without any fractionation, and CSQ2 was detected by Western blotting; this method avoided the >90% loss of CSQ2 occurring with usual fractionation procedures. Band intensities were compared against those for purified CSQ2 run on the same blots. Fidelity of quantification was verified by demonstrating that CSQ2 added to homogenates was detected with equal efficacy as purified CSQ2 alone. Ventricular tissue from sheep (n=8) contained 24±2 μmol CSQ2/kg wet wt. Total Ca2+ content of the ventricular tissue, measured by atomic absorption spectroscopy, was 430±20 μmol/kg (with SR Ca2+ likely<250 μmol/kg) and displayed a linear correlation with CSQ2 content, with gradient of ~10 Ca2+ per CSQ2. The large amount of CSQ2 bestows the SR with a high theoretical maximal Ca2+-binding capacity (~1 mmol Ca2+/kg ventricular tissue, assuming a maximum of ~40 Ca2+ per CSQ2) and would keep free [Ca2+] within the SR relatively low, energetically favoring Ca2+ uptake and reducing SR leak. In mice with CSQ2 ablated, histidine-rich Ca2+-binding protein was upregulated ~35% in ventricular tissue, possibly in compensation.  相似文献   

8.
We have examined the possible role of clathrin-coated vesicles (CVs) in the genesis of the sarcoplasmic reticulum (SR) in developing chick skeletal myotubes. Calsequestrin (CSQ) a luminal Ca2+ binding protein of the terminal SR cisternae, is contained within the vesicle lumen of skeletal muscle CVs in substantial amounts, approximately four molecules/CV. Employing 3-day cultures of chick skeletal myotubes we demonstrate that after a 30-min labeling with [35S]methionine and cysteine, radioactivity in CSQ remains high in the CVs 45 min later and then declines, while labeled CSQ in the SR continues to rise. No CSQ appears to be secreted. All of the CSQ in both the CVs and SR is sensitive to the activity of endoglycosidase H, and a significant fraction also binds to wheat germ agglutinin. Based on these results, we discuss the hypothesis that a selective CV-mediated pathway exists in developing skeletal muscle cells for the transport of CSQ from the early/intermediate Golgi apparatus to the SR.  相似文献   

9.
Calsequestrin (CSQ) is the low affinity, high capacity Ca(2+)-binding protein concentrated within specialized areas of the muscle fiber sarcoplasmic reticulum (a part of the ER) where it is believed to buffer large amounts of Ca2+. Upon activation of intracellular channels this Ca2+ pool is released, giving rise to the [Ca2+]i increases that sustain contraction. In order to investigate the ER retention and the functional role of the protein, L6 rat myoblasts were infected with a viral vector with or without the cDNA of chicken CSQ, and stable clones were investigated before and after differentiation to myotubes. In the undifferentiated L6 cells, expression of considerable amounts of heterologous CSQ occurred with no major changes of other ER components. Ca2+ release from the ER, induced by the peptide hormone vasopressin, remained however unchanged, and the same occurred when other treatments were given in sequence to deplete the ER and other intracellular stores: with the Ca2+ pump blocker, thapsigargin; and with the Ca2+ ionophore, ionomycin, followed by the Na+/H+ ionophore, monensin. The lack of effect of CSQ expression on the vasopressin-induced [Ca2+]i responses was explained by immunocytochemistry showing the heterologous protein to be localized not in the ER but in large vacuoles of acidic content, positive also for the lysosomal enzyme, cathepsin D, corresponding to a lysosomal subpopulation. After differentiation, all L6 cells expressed small amounts of homologous CSQ. In the infected cells the heterologous protein progressively decreased, yet the [Ca2+]i responses to vasopressin were now larger with respect to both control and undifferentiated cells. This change correlated with the drop of the vacuoles and with the accumulation of CSQ within the ER lumen, where a clustered distribution was observed as recently shown in developing muscle fibers. These results provide direct evidence for the contribution of CSQ, when appropriately retained, to the Ca2+ capacity of the rapidly exchanging, ER-located Ca2+ stores; and for the existence of specific mechanism(s) (that in L6 cells develop in the course of differentiation) for the ER retention of the protein. In the growing L6 myoblasts the Ca(2+)-binding protein appears in contrast to travel along the exocytic pathway, down to post-Golgi, lysosome-related vacuoles which, based on the lack of [Ca2+]i response to ionomycin- monensin, appear to be incompetent for Ca2+ accumulation.  相似文献   

10.
Calsequestrin (CSQ) is a high capacity Ca(2+)-binding protein present in the lumen of sarcoplasmic reticulum (SR) in striated muscle cells and has been shown to regulate the ryanodine receptor Ca(2+) release channel activity through interaction with other proteins present in the SR. Here we show that overexpression of wild-type CSQ or a CSQ mutant lacking the junction binding region (amino acids 86-191; Delta junc-CSQ) in mouse skeletal C2C12 myotube enhanced caffeine- and voltage-induced Ca(2+) release by increasing the Ca(2+) load in SR, whereas overexpression of a mutant CSQ lacking a Ca(2+) binding, aspartate-rich domain (amino acids 352-367; Delta asp-CSQ) showed the opposite effects. Depletion of SR Ca(2+) by thapsigargin initiated store-operated Ca(2+) entry (SOCE) in C2C12 myotubes. A large component of SOCE was inhibited by overexpression of wild-type CSQ or Delta junc-CSQ, whereas myotubes transfected with Delta asp-CSQ exhibited normal function of SOCE. These results indicate that the aspartate-rich segment of CSQ, under conditions of overexpression, can sustain structural interactions that interfere with the SOCE mechanism. Such retrograde activation mechanisms are possibly taking place at the junctional site of the SR.  相似文献   

11.
Ma J  Pan Z 《Cell calcium》2003,33(5-6):375-384
Store-operated Ca2+ entry represents an important mechanism for refilling of a depleted intracellular-reticulum Ca2+ store following sustained activation of the IP3 receptor or ryanodine receptor RyR/Ca2+ release channel in the endoplasmic/sarcoplasmic reticulum (ER/SR). Recent studies have demonstrated the existence of store-operated Ca2+ channel (SOC) in muscle cells, whose activation process appears to be coupled to conformational changes of the RyR. Regulation of the plasma membrane (PM)-resided SOC by the SR-located RyR requires an integrity of the junctional membrane structure between SR and PM. Proteins that interact with RyR or influence the Ca2+ buffering capacity in the ER or SR lumen also participate in the activation process of SOC. Calsequestrin (CSQ) and calreticulin (CRT) are SR/ER-resident proteins, with highly negative charged regions at the carboxyl-terminal end that exhibit high buffering capacity for luminal Ca2+. CSQ and CRT not only modulate the intracellular Ca2+ release process but also might provide retrograde signals to regulate the function of SOC. The functional interplay between CSQ, RyR and SOC may serve essential roles of Ca2+ signaling in muscle contraction and development. A tight link between the expression of CRT and operation of SOC exist in certain cancer cells, where the reduced sensitivity to apoptosis may correlate with the altered function of SOC.  相似文献   

12.
Enwrapment by membrane cisternae has emerged recently as a mechanism of envelopment for large enveloped DNA viruses, such as herpesviruses, poxviruses, and African swine fever (ASF) virus. For both ASF virus and the poxviruses, wrapping is a multistage process initiated by the recruitment of capsid proteins onto membrane cisternae of the endoplasmic reticulum (ER) or associated ER-Golgi intermediate membrane compartments. Capsid assembly induces progressive bending of membrane cisternae into the characteristic shape of viral particles, and envelopment provides virions with two membranes in one step. We have used biochemical assays for ASF virus capsid recruitment, assembly, and envelopment to define the cellular processes important for the enwrapment of viruses by membrane cisternae. Capsid assembly on the ER membrane, and envelopment by ER cisternae, were inhibited when cells were depleted of ATP or depleted of calcium by incubation with A23187 and EDTA or the ER calcium ATPase inhibitor, thapsigargin. Electron microscopy analysis showed that cells depleted of calcium were unable to assemble icosahedral particles. Instead, assembly sites contained crescent-shaped and bulbous structures and, in rare cases, empty closed five-sided particles. Interestingly, recruitment of the capsid protein from the cytosol onto the ER membrane did not require ATP or an intact ER calcium store. The results show that following recruitment of the virus capsid protein onto the ER membrane, subsequent stages of capsid assembly and enwrapment are dependent on ATP and are regulated by the calcium gradients present across the ER membrane cisternae.  相似文献   

13.
14.
To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins. FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments. Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins. Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes. The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT. The intermolecular DAT interactions were confirmed by co-immunoprecipitation. A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER. Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER. These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes.  相似文献   

15.
Infection of cells by picornaviruses leads to the generation of intracellular membrane vesicles. The expression of poliovirus (PV) 3A protein causes swelling of the endoplasmic reticulum (ER) and inhibition of protein trafficking between the ER and the Golgi apparatus. Here, we report that the nonstructural proteins of a second picornavirus, foot-and-mouth disease virus (FMDV), also perturb the secretory pathway. FMDV proteins 3A, 2B, 2C, and 2BC expressed alone in cells were recovered from crude membrane fractions, indicating membrane association. Immunofluorescence microscopy showed that 3A was located in a reticular structure and 2B was located in the ER, while 2C was located in both the ER and the bright punctate structures within the Golgi apparatus. 2BC gave punctate cytoplasmic staining and also caused accumulation of ER proteins in large vesicular structures located around the nuclei. The effect of the FMDV proteins on the trafficking of the vesicular stomatitis virus glycoprotein (G protein) from the ER to the cell surface was determined. Unlike its PV counterpart, the 3A protein of FMDV did not prevent trafficking of the G protein to the cell surface. Instead, surface expression of the G protein was blocked by 2BC, with retention of the G protein in a modified ER compartment staining for 2BC. The results suggest that the nonstructural proteins of different picornaviruses may vary in their ability to perturb the secretory pathway. Since FMDV 2BC can block the delivery of proteins to the cell surface, it may, as shown for PV 3A, play a role in immune evasion and contribute to the persistent infections observed in ruminants.  相似文献   

16.
Correct folding of a nascent polypeptide in the lumen of the endoplasmic reticulum (ER) into a three-dimensional conformation is a crucial step in the stability, intracellular trafficking, and targeting to the final destination of a protein. By transiently and stably expressing human-relevant mutants of Tamm-Horsfall protein in polarized Madin-Darby canine kidney cells, we show here that a cysteine-altering mutation in the evolutionally conserved cysteine-rich domain had more severe defects in ER exit and surface translocation and triggered more apoptosis than a cysteine-altering mutation outside the domain. Both mutants were able to specifically bind and trap the wild-type Tamm-Horsfall protein (THP) and prevent it from exiting the ER and translocating to the cell surface. This explains at least partly why in patients with THP-associated diseases there is a marked urinary reduction of both the mutant and the wild-type THP. Exposure of mutant-expressing cells to low temperature (30 °C), osmolytes (glycerol, trimethylamine N-oxide, and dimethyl sulfoxide), and the Ca(2+)-ATP inhibitor thapsigargin only slightly relieved ER retention and increased surface targeting of the mutants. In contrast, sodium 4-phenylbutyrate and probenecid, the latter a uricosuric drug used clinically to treat gout, markedly reduced ER retention of the mutants and increased their surface translocation and secretion into the culture media. The rescue of the THP mutants was associated with the restoration of the level and subcellular localization of cytosolic chaperone HSP70. Our results reveal intricate mechanistic details that may underlie THP-associated diseases and suggest that novel therapeutics enhancing the refolding of THP mutants may be of important value in therapy.  相似文献   

17.
We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.  相似文献   

18.
Reduced sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA2) contributes to the impaired cardiomyocyte Ca(2+) homeostasis observed in heart failure. We hypothesized that a reduction in SERCA2 also elicits myocardial ER/SR stress responses, including unfolded protein responses (UPR) and cardiomyocyte apoptosis, which may additionally contribute to the pathophysiology of this condition. Left ventricular myocardium from mice with cardiomyocyte-specific tamoxifen-inducible disruption of Serca2 (SERCA2 KO) was compared with aged-matched controls. In SERCA2 KO hearts, SERCA2 protein levels were markedly reduced to 2% of control values at 7 weeks following tamoxifen treatment. Serca2 disruption caused increased abundance of the ER stress-associated proteins CRT, GRP78, PERK, and eIF2α and increased phosphorylation of PERK and eIF2α, indicating UPR induction. Pro-apoptotic signaling was also activated in SERCA2 KO, as the abundance of CHOP, caspase 12, and Bax was increased. Indeed, TUNEL staining revealed an increased fraction of cardiomyocytes undergoing apoptosis in SERCA2 KO. ER-Tracker staining additionally revealed altered ER structure. These findings indicate that reduction in SERCA2 protein abundance is associated with marked ER/SR stress in cardiomyocytes, which induces UPR, apoptosis, and ER/SR structural alterations. This suggests that reduced SERCA2 abundance or function may contribute to the phenotype of heart failure also through induction of ER/SR stress responses.  相似文献   

19.
The cyclophilin homolog ninaA is required in the secretory pathway   总被引:38,自引:0,他引:38  
N J Colley  E K Baker  M A Stamnes  C S Zuker 《Cell》1991,67(2):255-263
In Drosophila, the major rhodopsin Rh1 is synthesized in endoplasmic reticulum (ER)-bound ribosomes of the R1-R6 photoreceptor cells and is then transported to the rhabdomeres where it functions in phototransduction. Mutations in the cyclophilin homolog ninaA lead to a 90% reduction in Rh1 opsin. Cyclophilins have been shown to be peptidyl-prolyl cis-trans isomerases and have been implicated in catalyzing protein folding. We now show that mutations in the ninaA gene severely inhibit opsin transport from the ER, leading to dramatic accumulations of ER cisternae in the photoreceptor cells. These results demonstrate that ninaA functions in the ER. Interestingly, ninaA and Rh1 also colocalize to secretory vesicles, suggesting that Rh1 may require ninaA as it travels through the distal compartments of the secretory pathway. These results are discussed in relation to the possible role of cyclophilins in protein folding and intracellular protein trafficking.  相似文献   

20.
The sarcoplasmic reticulum (SR) of skeletal muscle controls the contraction-relaxation cycle by raising and lowering the myoplasmic free-Ca2+ concentration. The coupling between excitation, i.e., depolarization of sarcolemma and transvers tubule (TT) and Ca2+ release from the terminal cisternae (TC) of SR takes place at the triad. The triad junction is formed by a specialized region of the TC, the junctional SR, and the TT. The molecular architecture and protein composition of the junctional SR are under active investigation. Since the junctional SR plays a central role in excitation-contraction coupling and Ca2+ release, some of its protein constituents are directly involved in these processes. The biochemical evidence supporting this contention is reviewed in this article.  相似文献   

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