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• W2065432039 abstract "Caveolin-3 is the principal structural protein of caveolae in striated muscle. Autosomal dominant limb-girdle muscular dystrophy (LGMD-1C) in humans is due to mutations (ΔTFT and Pro → Leu) within the CAV3 gene. We have shown that LGMD-1C mutations lead to formation of unstable aggregates of caveolin-3 that are retained intracellularly and are rapidly degraded. The mechanism by which LGMD-1C mutants of caveolin-3 are degraded remains unknown. Here, we show that LGMD-1C mutants of caveolin-3 undergo ubiquitination-proteasomal degradation. Treatment with proteasomal inhibitors (MG-132, MG-115, lactacystin, or proteasome inhibitor I), but not lysosomal inhibitors, prevented degradation of LGMD-1C caveolin-3 mutants. In the presence of MG-132, LGMD-1C caveolin-3 mutants accumulated within the endoplasmic reticulum and did not reach the plasma membrane. LGMD-1C mutants of caveolin-3 behave in a dominant negative fashion, causing intracellular retention and degradation of wild-type caveolin-3. Interestingly, in cells co-expressing wild-type and mutant forms of caveolin-3, MG-132 treatment rescued wild-type caveolin-3; wild-type caveolin-3 was not degraded and reached the plasma membrane. These results may have clinical implications for treatment of patients with LGMD-1C. Caveolin-3 is the principal structural protein of caveolae in striated muscle. Autosomal dominant limb-girdle muscular dystrophy (LGMD-1C) in humans is due to mutations (ΔTFT and Pro → Leu) within the CAV3 gene. We have shown that LGMD-1C mutations lead to formation of unstable aggregates of caveolin-3 that are retained intracellularly and are rapidly degraded. The mechanism by which LGMD-1C mutants of caveolin-3 are degraded remains unknown. Here, we show that LGMD-1C mutants of caveolin-3 undergo ubiquitination-proteasomal degradation. Treatment with proteasomal inhibitors (MG-132, MG-115, lactacystin, or proteasome inhibitor I), but not lysosomal inhibitors, prevented degradation of LGMD-1C caveolin-3 mutants. In the presence of MG-132, LGMD-1C caveolin-3 mutants accumulated within the endoplasmic reticulum and did not reach the plasma membrane. LGMD-1C mutants of caveolin-3 behave in a dominant negative fashion, causing intracellular retention and degradation of wild-type caveolin-3. Interestingly, in cells co-expressing wild-type and mutant forms of caveolin-3, MG-132 treatment rescued wild-type caveolin-3; wild-type caveolin-3 was not degraded and reached the plasma membrane. These results may have clinical implications for treatment of patients with LGMD-1C. limb-girdle muscular dystrophy monoclonal antibody polyclonal antibody phosphate-buffered saline polyacrylamide gel electrophoresis green fluorescent protein hemagglutinin 4-morpholineethanesulfonic acid wild type endoplasmic reticulum Caveolae are 50–100-nm vesicular invaginations of the plasma membrane (1Severs N.J. J. Cell Sci. 1988; 90: 341-348Crossref PubMed Google Scholar). It has been proposed that caveolae participate in vesicular trafficking events and signal transduction processes (2Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (585) Google Scholar, 3Couet J. Li S. Okamoto T. Scherer P.S. Lisanti M.P. Trends Cardiovasc. Med. 1997; 7: 103-110Crossref PubMed Scopus (111) Google Scholar, 4Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1336) Google Scholar, 5Engelman J.A. Zhang X.L. Galbiati F. Volonte D. Sotgia F. Pestell R.G. Minetti C. Scherer P.E. Okamoto T. Lisanti M.P. Am. J. Hum. Genet. 1998; 63: 1578-1587Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 6Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (917) Google Scholar, 7Razani B. Schlegel A. Lisanti M.P. J. Cell Sci. 2000; 113: 2103-2109Crossref PubMed Google Scholar). Caveolin, a 21–24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo (8Glenney Jr., J.R. J. Biol. Chem. 1989; 264: 20163-20166Abstract Full Text PDF PubMed Google Scholar, 9Glenney J.R. Soppet D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10517-10521Crossref PubMed Scopus (338) Google Scholar, 10Glenney J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (188) Google Scholar, 11Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1842) Google Scholar, 12Kurzchalia T. Dupree P. Parton R.G. Kellner R. Virta H. Lehnert M. Simons K. J. Cell Biol. 1992; 118: 1003-1014Crossref PubMed Scopus (462) Google Scholar). Caveolin is only the first member of a new gene family; as a consequence, caveolin has been re-termed caveolin-1 (13Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (486) Google Scholar). The mammalian caveolin gene family now consists of caveolins-1, -2, and -3 (4Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1336) Google Scholar, 5Engelman J.A. Zhang X.L. Galbiati F. Volonte D. Sotgia F. Pestell R.G. Minetti C. Scherer P.E. Okamoto T. Lisanti M.P. Am. J. Hum. Genet. 1998; 63: 1578-1587Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 13Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (486) Google Scholar, 14Parton R.G. Curr. Opin. Cell Biol. 1996; 8: 542-548Crossref PubMed Scopus (493) Google Scholar, 15Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). Caveolins 1 and 2 are co-expressed and form a hetero-oligomeric complex (16Scherer P.E. Lewis R.Y. Volonte D. Engelman J.A. Galbiati F. Couet J. Kohtz D.S. van Donselaar E. Peters P. Lisanti M.P. J. Biol. Chem. 1997; 272: 29337-29346Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar) in many cell types, with particularly high levels in adipocytes, whereas expression of caveolin-3 is muscle-specific and found in both cardiac and skeletal muscle (17Song K.S. Scherer P.E. Tang Z. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). The expression of caveolin-3 is induced during the differentiation of skeletal myoblasts, and caveolin-3 is localized to the muscle cell plasma membrane (sarcolemma) where it forms a complex with dystrophin and its associated glycoproteins (17Song K.S. Scherer P.E. Tang Z. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). However, under certain conditions caveolin-3 can be physically separated from the dystrophin complex (18Crosbie R.H. Yamada H. Venzke D.P. Lisanti M.P. Campbell K.P. FEBS Lett. 1998; 427: 279-282Crossref PubMed Scopus (73) Google Scholar). This indicates that although caveolin-3 is dystrophin-associated, it is not absolutely required for the biogenesis of the dystrophin complex (18Crosbie R.H. Yamada H. Venzke D.P. Lisanti M.P. Campbell K.P. FEBS Lett. 1998; 427: 279-282Crossref PubMed Scopus (73) Google Scholar). It has been proposed that caveolin family members function as scaffolding proteins (19Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (472) Google Scholar) to organize and concentrate specific lipids (cholesterol and glycosphingolipids (20Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 21Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (760) Google Scholar, 22Fra A.M. Masserini M. Palestini P. Sonnino S. Simons K. FEBS Lett. 1995; 375: 11-14Crossref PubMed Scopus (159) Google Scholar)) and lipid-modified signaling molecules (Src-like kinases, Ha-Ras, endothelial nitric-oxide synthase, and G-proteins (20Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 23Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Crossref PubMed Scopus (554) Google Scholar, 24Song K.S. Li S. Okamoto T. Quilliam L. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar, 25Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar, 26Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 27Garcia-Cardena G. Oh P. Liu J. Schnitzer J.E. Sessa W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (572) Google Scholar)) within caveolae membranes. These caveolin family members all share three characteristic properties: (i) detergent insolubility at low temperatures; (ii) self-oligomerization; and (iii) incorporation into low density Triton-insoluble membrane fractions that are enriched in caveolae-membranes (28Song K.S. Tang Z.-L. Li S. Lisanti M.P. J. Biol. Chem. 1997; 272: 4398-4403Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The specialized lipid composition of caveolae is thought to convey resistance of this membrane domain to detergent solubilization by Triton X-100 (at low temperatures) (29Sargiacomo M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (855) Google Scholar, 30Lisanti M.P. Tang Z.-L. Sargiacomo M. J. Cell Biol. 1993; 123: 595-604Crossref PubMed Scopus (158) Google Scholar, 31Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski- Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (806) Google Scholar, 32Scherer P.E. Lisanti M.P. Baldini G. Sargiacomo M. Corley-Mastick C. Lodish H.F. J. Cell Biol. 1994; 127: 1233-1243Crossref PubMed Scopus (349) Google Scholar, 33Chang W.J. Ying Y. Rothberg K. Hooper N. Turner A. Gambliel H. De Gunzburg J. Mumby S. Gilman A. Anderson R.G.W. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (308) Google Scholar, 34Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (633) Google Scholar, 35Schnitzer J.E. Oh P. Jacobson B.S. Dvorak A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1759-1763Crossref PubMed Scopus (227) Google Scholar). This property appears to be unique to caveolae membranes. For example, when intact cells were fixed in paraformaldehyde and extracted with Triton X-100 and then examined by electron microscopy, the insoluble membranes that remained were found to be caveolae (36Moldovan N. Heltianu C. Simionescu N. Simionescu M. Exp. Cell Res. 1995; 219: 309-313Crossref PubMed Scopus (32) Google Scholar). Caveolin-3 is most closely related to caveolin-1 based on protein sequence homology; caveolin-1 and caveolin-3 are ∼65% identical and ∼85% similar (see Tang et al. (15Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar) for an alignment). However, caveolin-3 mRNA is expressed predominantly in muscle tissue types (skeletal muscle, diaphragm, and heart) (15Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). Identification of a muscle-specific member of the caveolin gene family has implications for understanding the role of caveolins in different muscle cell types (smooth, cardiac, and skeletal), as previous morphological studies have demonstrated that caveolae are abundant in these cells. A number of studies have highlighted the importance of caveolae and caveolins in the pathogenesis of Duchenne's muscular dystrophy. More specifically, dystrophin has been localized to plasma membrane caveolae in smooth muscle cells using immuno-EM techniques (37North A.J. Galazkiewicz B. Byers T.J. Glenney J.R. Small J.V. J. Cell Biol. 1993; 120: 1159-1167Crossref PubMed Scopus (141) Google Scholar), and skeletal muscle caveolae undergo characteristic changes in their size and distribution in patients with Duchenne's muscular dystrophy but not in other forms of neuronally based muscular dystrophies examined (38Bonilla E. Fishbeck K. Schotland D. Am. J. Pathol. 1981; 104: 167-173PubMed Google Scholar). This indicates that muscle cell caveolae may play an important role in muscle membrane biology. In collaboration with Minetti and colleagues (39Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonté D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (483) Google Scholar), we have recently identified an autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C)1 in two Italian families that is due to a deficiency in caveolin-3 expression. Analysis of their genomic DNA reveals two distinct mutations in the caveolin-3 gene as follows: (i) a 9-base pair micro-deletion that removes the sequence TFT from the caveolin-scaffolding domain, and (ii) a mis-sense mutation that changes a proline to a leucine (Pro → Leu) in the transmembrane domain (39Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonté D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (483) Google Scholar). Both mutations lead to a loss of ∼90–95% of caveolin-3 protein expression. Characterization of CAV3 lesions in these LGMD-1C families is instructive. Twelve amino acid residues are absolutely conserved in all three human caveolins as well as the two Caenorhabditis elegans caveolins (40Tang Z. Okamoto T. Boontrakulpoontawee P. Katada T. Otsuka A.J. Lisanti M.P. J. Biol. Chem. 1997; 272: 2437-2445Crossref PubMed Scopus (80) Google Scholar), and the LGMD-1C mutants map to 2 of these 12 invariant residues, Pro104 and Phe64 (39Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonté D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (483) Google Scholar). In addition, alanine scanning mutagenesis of a peptide encoding the caveolin-scaffolding domain reveals that the FTV(T/S) sequence in caveolins 1 and 3 is important for recognition of caveolin binding, signaling proteins (41Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar); the FT di-peptide of the FTVT/S sequence is deleted in one LGMD-1C family. This finding provides genetic evidence that this region of the caveolin-scaffolding domain is criticalin vivo. How do mutations in these invariant residues give rise to LGMD-1C? By using heterologous expression in NIH 3T3 cells, we demonstrated that down-regulation of the caveolin-3 protein in LGMD-1C may reflect the targeting of mis-folded caveolin-3 oligomers for degradation (42Galbiati F. Volonté D. Minetti C. Chu J.B. Lisanti M.P. J. Biol. Chem. 1999; 274: 25632-25641Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Both LGMD-1C mutants of caveolin-3 form unstable aggregates that are retained within the Golgi complex (42Galbiati F. Volonté D. Minetti C. Chu J.B. Lisanti M.P. J. Biol. Chem. 1999; 274: 25632-25641Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Consistent with its autosomal dominant form of genetic transmission (39Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonté D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (483) Google Scholar), both LGMD-1C caveolin-3 mutants cause retention of wild-type caveolin-3 in the Golgi compartment and induce the degradation of wild-type caveolin-3 (42Galbiati F. Volonté D. Minetti C. Chu J.B. Lisanti M.P. J. Biol. Chem. 1999; 274: 25632-25641Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). These data provide a molecular explanation for caveolin-3 down-regulation in patients with this form of limb-girdle muscular dystrophy. However, the mechanism(s) by which LGMD-1C mutants of caveolin-3 are degraded remain unknown. This may have important implications for the treatment of LGMD-1C patients, as these patients still retain one wild-type allele of caveolin-3. Here, we show that LGMD-1C mutants of caveolin-3 undergo ubiquitination/proteasomal degradation. Treatment with proteasomal inhibitors (MG-132, MG-115, lactacystin, or proteasome inhibitor I), but not lysosomal inhibitors, prevented degradation of LGMD-1C caveolin-3 mutants. Consistent with the idea that LGMD-1C mutants are mis-folded, conditions that facilitate protein folding (10% glycerol or 30 °C) also prevented degradation of LGMD-1C caveolin-3 mutants. Interestingly, in cells co-expressing wild-type and mutant forms of caveolin-3, MG-132 treatment rescued wild-type caveolin-3; under these conditions, wild-type caveolin-3 was not degraded and reached the plasma membrane. Thus, treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutants and rescues wild-type caveolin-3. This is the first demonstration that proteasomal degradation may be involved in a form of muscular dystrophy in humans. Antibodies and their sources were as follow: anti-caveolin-3 IgG (mAb 26 (17), gift of Dr. Roberto Campos-Gonzalez, BD-Transduction Laboratories); anti-caveolin-1 IgG (mAb 2297; (43Scherer P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar); gift of Dr. Roberto Campos-Gonzalez, BD-Transduction Laboratories); anti-calnexin IgG (pAb, gift of Dr. Peter Arvan, Albert Einstein College of Medicine); anti-Myc IgG (pAb, Santa Cruz Biotechnology, Santa Cruz, CA). Proteasome inhibitors (MG-132, MG-115, lactacystin, and proteasome inhibitor I) were from Calbiochem. Untagged LGMD-1C caveolin-3 mutants were generated as we described previously (42Galbiati F. Volonté D. Minetti C. Chu J.B. Lisanti M.P. J. Biol. Chem. 1999; 274: 25632-25641Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The cDNA encoding HA-tagged ubiquitin was as described previously (44Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (845) Google Scholar, 45Bregman D.B. Halaban R. van Gool A.J. Henning K.A. Friedberg E.C. Warren S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11586-11590Crossref PubMed Scopus (262) Google Scholar, 46Ratner J.N. Balasubramanian B. Corden J. Warren S.L. Bregman D.B. J. Biol. Chem. 1998; 273: 5184-5189Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). All other biochemicals used were of the highest purity available and were obtained from regular commercial sources. NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with glutamine, antibiotics (penicillin and streptomycin), and 10% donor bovine calf serum. Cells (∼30–50% confluent) were transfected using a modified calcium phosphate precipitation method. Thirty six hours post-transfection, cells were treated for 16 h with vehicle alone (Me2SO) or with one of the following proteasome inhibitors: MG-132 (5 or 10 μm), MG-115 (5 μm), lactacystin (20 μm), or proteasome inhibitor I (20 μm). Proteasome inhibitors were dissolved in Me2SO. Transfected NIH 3T3 cells were scraped in 2 ml of Mes-buffered saline containing 1% (v/v) Triton X-100. Homogenization was carried out with 10 strokes of a loose fitting Dounce homogenizer. The homogenate was adjusted to 40% sucrose by the addition of 2 ml of 80% sucrose prepared in Mes-buffered saline and placed at the bottom of an ultracentrifuge tube. A 5–30% linear sucrose gradient was formed above the homogenate and centrifuged at 39,000 rpm for 16–20 h in a SW41 rotor (Beckman Instruments). A light-scattering band confined to the 15–20% sucrose region was observed that contained endogenous caveolin-1 but excluded most of other cellular proteins. From the top of each gradient, 1-ml gradient fractions were collected to yield a total of 12 fractions. An equal amount of protein from each gradient fraction was separated by SDS-PAGE and subjected to immunoblot analysis. Cellular proteins were resolved by SDS-PAGE (12.5% acrylamide) and transferred to nitrocellulose membranes. Blots were incubated for 2 h in TBST (10 mmTris-HCl, pH 8.0, 150 mm NaCl, 0.2% Tween 20) containing 2% powdered skim milk and 1% bovine serum albumin. After three washes with TBST, membranes were incubated for 2 h with the primary antibody (∼1000-fold diluted in TBST) and for 1 h with horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG (∼5000-fold diluted). Proteins were detected using an ECL detection kit (Amersham Pharmacia Biotech). NIH 3T3 cells grown on glass coverslips were washed three times with PBS and fixed for 30 min at room temperature with 2% paraformaldehyde in PBS. Fixed cells were rinsed with PBS and permeabilized with 0.1% Triton X-100, 0.2% bovine serum albumin for 10 min. Then cells were treated with 25 mm NH4Cl in PBS for 10 min at room temperature to quench free aldehyde groups. Cells were rinsed with PBS and incubated with the primary antibodies for 1 h at room temperature (∼1000-fold diluted in PBS with 0.1% Triton X-100, 0.2% bovine serum albumin). After three washes with PBS (10 min each), cells were incubated with the secondary antibody for 1 h at room temperature, lissamine rhodamine B sulfonyl chloride-conjugated goat anti-rabbit antibody (5 μg/ml)/fluorescein isothiocyanate-conjugated goat anti-mouse antibody (5 μg/ml). Finally, cells were washed three times with PBS (10 min each wash), and slides were mounted with slow-fade anti-fade reagent (Molecular Probes, Inc., Eugene, OR) and observed under a Bio-Rad MR 600 confocal microscope. Cells were washed twice with PBS and lysed 30 min at 4 °C in a buffer containing 10 mm Tris, pH 8.0, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 60 mm octyl glucoside. Samples were pre-cleared for 1 h at 4 °C using protein A-Sepharose (20 μl, slurry 1:1) and subjected to overnight immunoprecipitation at 4 °C using anti-caveolin-3 antibody (10 μl, mAb) and protein A-Sepharose (30 μl, slurry 1:1). After three washes with the immunoprecipitation buffer, samples were separated by SDS-PAGE (12.5% acrylamide) and transferred to nitrocellulose. Blots were then probed with anti-HA tag antibody (pAb; Santa Cruz Biotechnology, Inc.). NIH 3T3 cells were co-transfected with C-terminally Myc-tagged WT caveolin-3 and three different N-terminally GFP-tagged caveolin-3 fusions as we previously described (42Galbiati F. Volonté D. Minetti C. Chu J.B. Lisanti M.P. J. Biol. Chem. 1999; 274: 25632-25641Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Myc-tagged caveolin-3 was visualized using pAb A-14 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) that is directed against the Myc epitope (EQKLISEEDLN). GFP-tagged caveolin-3 was detected as we previously described (42Galbiati F. Volonté D. Minetti C. Chu J.B. Lisanti M.P. J. Biol. Chem. 1999; 274: 25632-25641Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 47Volonté D. Galbiati F. Lisanti M.P. FEBS Lett. 1999; 445: 431-439Crossref PubMed Scopus (60) Google Scholar). Wild-type caveolin-3 and caveolin-3 mutants (Pro → Leu or ΔTFT) were transiently expressed in NIH 3T3 cells. Twenty four hours post-transfection, cells were cultured for an additional 16 h at low temperature (30 °C) or with normal medium supplemented with 10% (v/v) glycerol at 37 °C; the expression of caveolin-3 was then assessed by immunoblotting. Control cells were cultured for the same amount of time at 37 °C and in the absence of glycerol. Autosomal dominant limb-girdle muscular dystrophy (LGMD-1C) in humans is due to mutations (ΔTFT and Pro → Leu) within the CAV3 gene (39Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonté D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (483) Google Scholar). We have shown that LGMD-1C mutations lead to formation of unstable aggregates of caveolin-3 that are retained intracellularly, and they have a dramatically shortened half-life of ∼45–60 min (42Galbiati F. Volonté D. Minetti C. Chu J.B. Lisanti M.P. J. Biol. Chem. 1999; 274: 25632-25641Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). We previously generated and characterized the same two mutations in wild-type caveolin-3 that are seen in patients with limb-girdle muscular dystrophy 1C (LGMD-1C) (42Galbiati F. Volonté D. Minetti C. Chu J.B. Lisanti M.P. J. Biol. Chem. 1999; 274: 25632-25641Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Here, we have begun to evaluate the hypothesis that the lower protein expression of the LGMD-1C caveolin-3 mutants was due to protein degradation occurring through the proteasome pathway. For this purpose, we analyzed the effect of proteasome inhibitor treatment on the expression of the LGMD-1C caveolin-3 mutants. We transiently transfected NIH 3T3 cells with wild-type caveolin-3 or the corresponding LGMD-1C mutants (Pro → Leu and ΔTFT) and assessed their expression by Western blot analysis with a specific caveolin-3 monoclonal antibody probe (mAb 26) (Fig.1). Note that both mutant forms are expressed at significantly lower levels than achieved with wild-type caveolin-3. However, treatment for 16 h with MG-132 (10 μm) was sufficient to rescue the expression of both LGMD-1C caveolin-3 mutants.Figure 1Treatment with MG-132, a proteasome inhibitor, blocks the degradation of the LGMD-1C mutants of caveolin-3. Wild-type caveolin-3 (Cav-3) and caveolin-3 mutants (Pro → Leu or ΔTFT) were transiently expressed in NIH 3T3 cells. After treatment for 16 h with the vehicle alone (Me2SO, DMSO) or with MG-132 (10 μm), the expression of caveolin-3 was assessed by immunoblotting with a specific caveolin-3 monoclonal antibody probe (17Song K.S. Scherer P.E. Tang Z. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar).View Large Image Figure ViewerDownload (PPT) The ability of the proteasome inhibitor MG-132 to rescue the expression of the LGMD-1C caveolin-3 mutants became apparent at 1 μmand was maximal at a concentration of 5 μm (Fig.2). Interestingly, endogenous caveolin-1 expression was not modified by proteasome inhibitor treatment. These results suggest that the degradation is specific for LGMD-1C caveolin-3 mutants (Pro → Leu and ΔTFT) (Fig. 2), and occurs through the proteasome pathway. To assess whether other proteasomal inhibitors are capable of preventing the degradation of the LGMD-1C mutants of caveolin-3, we next treated transiently transfected NIH 3T3 cells for 16 h with several different proteasome inhibitors as follows: MG-132 (5 μm), MG-115 (5 μm), lactacystin (20 μm), or proteasome inhibitor I (20 μm). Fig. 3 A shows that the expression of the LGMD-1C mutants of caveolin-3 is significantly increased by all of the proteasomal inhibitors that we tested. Proteins that are degraded by proteasome system are often first conjugated to multiple copies of the small protein ubiquitin through isopeptide linkages (44Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (845) Google Scholar, 45Bregman D.B. Halaban R. van Gool A.J. Henning K.A. Friedberg E.C. Warren S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11586-11590Crossref PubMed Scopus (262) Google Scholar, 46Ratner J.N. Balasubramanian B. Corden J. Warren S.L. Bregman D.B. J. Biol. Chem. 1998; 273: 5184-5189Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Thus, inhibition of the proteasome system results in the accumulation of ubiquitinated forms of proteins normally degraded by the proteasome. To evaluate whether LGMD-1C caveolin-3 mutants are ubiquitinated prior to proteasomal degradation, we co-transfected these caveolin-3 mutants (Pro → Leu or ΔTFT) with an HA-tagged form of ubiquitin. Cells were then treated for 16 h with either vehicle alone (Me2SO) or MG-132 (5 μm) and subjected to immunoprecipitation with a caveolin-3-specific mouse mAb (clone 26). These immunoprecipitates were then analyzed by Western blotting using a rabbit pAb directed against the HA tag. Fig. 3 B shows that inhibition of the proteasomal machinery by treatment with MG-132 results in the accumulation of multiple ubiquitinated forms of LGMD-1C caveolin-3 mutants. However, in the absence of MG-132, no ubiquitinated forms of caveolin-3 were detected. Interestingly, the human caveolin-3 protein contains 9 lysine r" @default.
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