Immunological evidence for eight spans in the membrane domain of 3- hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum

doi:10.1083/jcb.117.5.959

This Article

	Full Text (PDF, 5811K)
	Alert me when this article is cited
	Citation Map

Services

	Email this article
	Similar articles in this journal
	Similar articles in PubMed
	Alert me to new content in the JCB
	Download to citation manager

Citing Articles

	Citing Articles via HighWire
	Citing Articles via CrossRef
	Citing Articles via Google Scholar

Google Scholar

	Articles by Roitelman, J.
	Articles by Simoni, R. D.
	Search for Related Content

PubMed

	PubMed Citation
	Articles by Roitelman, J.
	Articles by Simoni, R. D.


Social Bookmarking

	What's this?

ARTICLES

Immunological evidence for eight spans in the membrane domain of 3- hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum

J Roitelman, EH Olender, S Bar-Nun, WA Dunn Jr and RD Simoni
Department of Biological Sciences, Stanford University, California 94305.

We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. Finer- Moore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.
Add to CiteULike CiteULike Add to Complore Complore Add to Connotea Connotea Add to Del.icio.us Del.icio.us Add to Digg Digg Facebook Add to Reddit Reddit Add to Technorati Technorati Twitter What's this?

This article has been cited by other articles:

Nguyen, A. D., Lee, S. H., DeBose-Boyd, R. A. (2009). Insig-mediated, Sterol-accelerated Degradation of the Membrane Domain of Hamster 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase in Insect Cells. J. Biol. Chem. 284: 26778-26788 [Abstract] [Full Text]
Leichner, G. S., Avner, R., Harats, D., Roitelman, J. (2009). Dislocation of HMG-CoA Reductase and Insig-1, Two Polytopic Endoplasmic Reticulum Proteins, En Route to Proteasomal Degradation. Mol. Biol. Cell 20: 3330-3341 [Abstract] [Full Text]
Brown, M. S., Goldstein, J. L. (2009). Cholesterol feedback: from Schoenheimer's bottle to Scap's MELADL. J. Lipid Res. 50: S15-S27 [Abstract] [Full Text]
Nguyen, A. D., McDonald, J. G., Bruick, R. K., DeBose-Boyd, R. A. (2007). Hypoxia Stimulates Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase through Accumulation of Lanosterol and Hypoxia-Inducible Factor-mediated Induction of Insigs. J. Biol. Chem. 282: 27436-27446 [Abstract] [Full Text]
Lee, P. C. W., Liu, P., Li, W.-P., DeBose-Boyd, R. A. (2007). Amplification of the gene for SCAP, coupled with Insig-1 deficiency, confers sterol resistance in mutant Chinese hamster ovary cells. J. Lipid Res. 48: 1944-1954 [Abstract] [Full Text]
Lee, P. C. W., Nguyen, A. D., DeBose-Boyd, R. A. (2007). Mutations within the membrane domain of HMG-CoA reductase confer resistance to sterol-accelerated degradation. J. Lipid Res. 48: 318-327 [Abstract] [Full Text]
Song, B.-L., DeBose-Boyd, R. A. (2006). Insig-dependent Ubiquitination and Degradation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Stimulated by {delta}- and {gamma}-Tocotrienols. J. Biol. Chem. 281: 25054-25061 [Abstract] [Full Text]
Almsherqi, Z. A., Kohlwein, S. D., Deng, Y. (2006). Cubic membranes: a legend beyond the Flatland* of cell membrane organization. JCB 173: 839-844 [Abstract] [Full Text]
Lee, P. C. W., Sever, N., DeBose-Boyd, R. A. (2005). Isolation of Sterol-resistant Chinese Hamster Ovary Cells with Genetic Deficiencies in Both Insig-1 and Insig-2. J. Biol. Chem. 280: 25242-25249 [Abstract] [Full Text]
Sever, N., Lee, P. C. W., Song, B.-L., Rawson, R. B., DeBose-Boyd, R. A. (2004). Isolation of Mutant Cells Lacking Insig-1 through Selection with SR-12813, an Agent That Stimulates Degradation of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase. J. Biol. Chem. 279: 43136-43147 [Abstract] [Full Text]
Doolman, R., Leichner, G. S., Avner, R., Roitelman, J. (2004). Ubiquitin Is Conjugated by Membrane Ubiquitin Ligase to Three Sites, including the N Terminus, in Transmembrane Region of Mammalian 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase: IMPLICATIONS FOR STEROL-REGULATED ENZYME DEGRADATION. J. Biol. Chem. 279: 38184-38193 [Abstract] [Full Text]
Song, B.-L., DeBose-Boyd, R. A. (2004). Ubiquitination of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Permeabilized Cells Mediated by Cytosolic E1 and a Putative Membrane-bound Ubiquitin Ligase. J. Biol. Chem. 279: 28798-28806 [Abstract] [Full Text]
Roitelman, J., Masson, D., Avner, R., Ammon-Zufferey, C., Perez, A., Guyon-Gellin, Y., Bentzen, C. L., Niesor, E. J. (2004). Apomine, a Novel Hypocholesterolemic Agent, Accelerates Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase and Stimulates Low Density Lipoprotein Receptor Activity. J. Biol. Chem. 279: 6465-6473 [Abstract] [Full Text]
Kikkert, M., Doolman, R., Dai, M., Avner, R., Hassink, G., van Voorden, S., Thanedar, S., Roitelman, J., Chau, V., Wiertz, E. (2004). Human HRD1 Is an E3 Ubiquitin Ligase Involved in Degradation of Proteins from the Endoplasmic Reticulum. J. Biol. Chem. 279: 3525-3534 [Abstract] [Full Text]
Sever, N., Song, B.-L., Yabe, D., Goldstein, J. L., Brown, M. S., DeBose-Boyd, R. A. (2003). Insig-dependent Ubiquitination and Degradation of Mammalian 3-Hydroxy-3-methylglutaryl-CoA Reductase Stimulated by Sterols and Geranylgeraniol. J. Biol. Chem. 278: 52479-52490 [Abstract] [Full Text]
Yasuda, S., Nishijima, M., Hanada, K. (2003). Localization, Topology, and Function of the LCB1 Subunit of Serine Palmitoyltransferase in Mammalian Cells. J. Biol. Chem. 278: 4176-4183 [Abstract] [Full Text]
Peffley, D. M., Gayen, A. K. (2003). Plant-Derived Monoterpenes Suppress Hamster Kidney Cell 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Synthesis at the Post-Transcriptional Level. J. Nutr. 133: 38-44 [Abstract] [Full Text]
Breitling, R., Krisans, S. K. (2002). A second gene for peroxisomal HMG-CoA reductase? A genomic reassessment. J. Lipid Res. 43: 2031-2036 [Abstract] [Full Text]
Gonzalez-Baro, M. R., Granger, D. A., Coleman, R. A. (2001). Mitochondrial Glycerol Phosphate Acyltransferase Contains Two Transmembrane Domains with the Active Site in the N-terminal Domain Facing the Cytosol. J. Biol. Chem. 276: 43182-43188 [Abstract] [Full Text]
Isaac, C., Pollard, J. W., Meier, U. T. (2001). Intranuclear endoplasmic reticulum induced by Nopp140 mimics the nucleolar channel system of human endometrium. J. Cell Sci. 114: 4253-4264 [Abstract] [Full Text]
Ravid, T., Avner, R., Polak-Charcon, S., Faust, J. R., Roitelman, J. (1999). Impaired Regulation of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase Degradation in Lovastatin-resistant Cells. J. Biol. Chem. 274: 29341-29351 [Abstract] [Full Text]
Lin, S., Cheng, D., Liu, M.-S., Chen, J., Chang, T.-Y. (1999). Human Acyl-CoA:Cholesterol Acyltransferase-1 in the Endoplasmic Reticulum Contains Seven Transmembrane Domains. J. Biol. Chem. 274: 23276-23285 [Abstract] [Full Text]
Moriyama, T., Sather, S. K., McGee, T. P., Simoni, R. D. (1998). Degradation of HMG-CoA Reductase in Vitro. CLEAVAGE IN THE MEMBRANE DOMAIN BY A MEMBRANE-BOUND CYSTEINE PROTEASE. J. Biol. Chem. 273: 22037-22043 [Abstract] [Full Text]
Nohturfft, A., Brown, M. S., Goldstein, J. L. (1998). Topology of SREBP Cleavage-activating Protein, a Polytopic Membrane Protein with a Sterol-sensing Domain. J. Biol. Chem. 273: 17243-17250 [Abstract] [Full Text]
Engfelt, W. H., Shackelford, J. E., Aboushadi, N., Jessani, N., Masuda, K., Paton, V. G., Keller, G.-A., Krisans, S. K. (1997). Characterization of UT2 Cells. THE INDUCTION OF PEROXISOMAL 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE. J. Biol. Chem. 272: 24579-24587 [Abstract] [Full Text]
Kim, T.-W., Pettingell, W. H., Hallmark, O. G., Moir, R. D., Wasco, W., Tanzi, R. E. (1997). Endoproteolytic Cleavage and Proteasomal Degradation of Presenilin 2in Transfected Cells. J. Biol. Chem. 272: 11006-11010 [Abstract] [Full Text]
Beachy, P.A., Cooper, M.K., Young, K.E., von Kessler, D.P., Park, W.-J., Hall, T.M. T., Leahy, D.J., Porter, J.A. (1997). Multiple Roles of Cholesterol in Hedgehog Protein Biogenesis and Signaling. Cold Spring Harb Symp Quant Biol 62: 191-204 [Abstract]
Dan, N., Middleton, R. B., Lehrman, M. A. (1996). Hamster UDP-N-Acetylglucosamine:Dolichol-P N-Acetylglucosamine-1-P Transferase Has Multiple Transmembrane Spans and a Critical Cytosolic Loop. J. Biol. Chem. 271: 30717-30724 [Abstract] [Full Text]
McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., Simoni, R. D. (1996). Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Endoplasmic Reticulum Membranes Is Accelerated as a Result of Increased Susceptibility to Proteolysis. J. Biol. Chem. 271: 25630-25638 [Abstract] [Full Text]
Denbow, C. J., L�ng, S., Cramer, C. L. (1996). The N-terminal Domain of Tomato 3-Hydroxy-3-methylglutaryl-CoA Reductases. J. Biol. Chem. 271: 9710-9715 [Abstract] [Full Text]
Meigs, T. E., Roseman, D. S., Simoni, R. D. (1996). Regulation of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase Degradation by the Nonsterol Mevalonate Metabolite Farnesol in Vivo. J. Biol. Chem. 271: 7916-7922 [Abstract] [Full Text]
Yamamoto, A, Masaki, R, Tashiro, Y (1996). Formation of crystalloid endoplasmic reticulum in COS cells upon overexpression of microsomal aldehyde dehydrogenase by cDNA transfection. J. Cell Sci. 109: 1727-1738 [Abstract]
Chang, C. C. Y., Chen, J., Thomas, M. A., Cheng, D., Priore, V. A. D., Newton, R. S., Pape, M. E., Chang, T.-Y. (1995). Regulation and Immunolocalization of Acyl-Coenzyme A:Cholesterol Acyltransferase in Mammalian Cells as Studied with Specific Antibodies. J. Biol. Chem. 270: 29532-29540 [Abstract] [Full Text]
Kumagai, H., Chun, K. T., Simoni, R. D. (1995). Molecular Dissection of the Role of the Membrane Domain in the Regulated Degradation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase. J. Biol. Chem. 270: 19107-19113 [Abstract] [Full Text]
Lawrence, C., Rodwell, V., Stauffacher, C. (1995). Crystal structure of Pseudomonas mevalonii HMG-CoA reductase at 3.0 angstrom resolution. Science 268: 1758-1762 [Abstract]
Lecureux, L., Wattenberg, B. (1994). The regulated degradation of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase reporter construct occurs in the endoplasmic reticulum. J. Cell Sci. 107: 2635-2642 [Abstract]
Ravid, T., Doolman, R., Avner, R., Harats, D., Roitelman, J. (2000). The Ubiquitin-Proteasome Pathway Mediates the Regulated Degradation of Mammalian 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase. J. Biol. Chem. 275: 35840-35847 [Abstract] [Full Text]
Hao, M., Lin, S. X., Karylowski, O. J., Wustner, D., McGraw, T. E., Maxfield, F. R. (2002). Vesicular and Non-vesicular Sterol Transport in Living Cells. THE ENDOCYTIC RECYCLING COMPARTMENT IS A MAJOR STEROL STORAGE ORGANELLE. J. Biol. Chem. 277: 609-617 [Abstract] [Full Text]