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Basic-helix-loop-helix transcription factor and sterol sensor in a single membrane-bound molecule
Cell, Vol. 77, 17-19, April 8, 1994, Copyright 0 1994 by Cell Press
Basic-Helix-Loop-Helix Transcription Factor and Sterol Sensor in a Single Membrane-Bound Molecule Gregory P. Gaslc Cell Press 50 Church Street Cambridge, Massachusetts 02139
Cholesterol and closely related sterols are present in the membranes of most eukaryoticcells, although they are not uniformly distributed among the different cell membrane compartments. In vertebrates, most of the cholesterol synthesis takes place in the liver, where a small portion is incorporated into the membranes of the hepatocytes and the remainder is exported as bile acids or cholesterol esters. The bile acids, stored in the gallbladder as salts, are released in the small intestine to aid with lipid digestion. Cholesterol esters are carried by plasma lipoproteins to tissues to be stored or consumed. Animal cells can obtain their cholesterol endogenously by synthesis from acetylcoenzyme A (CoA) through mevalonate or exogenously by receptor-mediated endocytosis of plasma low density lipoprotein (LDL). These cells must balance the internal and external sources of cholesterol to sustain the synthesis of mevalonate, a important precursor for the isoprenoid building blocks that are required to make sterols, electron transport moieties, glycoproteins, and farnesylated proteins, while avoiding sterol over accumulation (Goldstein and Brown, 1990). This balance is achieved through feedback regulation of the LDL receptor and through at least two consecutive enzymes in mevalonate synthesis, 3-hydroxy-3-methylglutaryl CoA (HMG CoA) synthase and HMG CoA reductase. For HMG CoA reductase, sterols exercise both transcriptional control and posttranslational control (Gil et al., 19&S), promoting the increased degradation of the enzyme in the endoplasmic reticulum (ER). A 10 bp sequence in the 5’flanking region, designated sterol regulatory element 1 (SRE-l), controls LDL receptor transcription such that when cellular sterol stores are expended, the gene is transcribed, receptor biosynthesis increases, and LDL uptake through receptor-mediated endocytosis increases (Gold stein and Brown, 1990). Likewise, when cellular sterol stores are replenished, SRE-1 activity is abolished and LDL receptor transcription decreases. SRE-1 is a conditional positive element that enhances LDL receptor and HMG CoA synthase gene transcription in the absence but not presence of sterols. The cDNAs coding for two proteins, SRE-binding proteins 1 and 2 (SREBP-1 and SREBP-P), each of which binds SRE-1 with a nucleotide specificity that is in perfect agreement with that required for sterol-regulated transcription (Wang et al., 1993), have been characterized (Yokoyama et al., 1993; Hua et al., 1993). Both contain a sequence (amino acid residues 324-394 of SREBP-1) that conforms to the consensus sequence of the basic-helixloop-helix-leucine zipper family of transcription factors (Murre and Baltimore, 1992; Pabo and Sauer, 1992). HOWever, there is an interesting contrast between the 125 kd
Minireview
protein that is produced when these cDNAs are expressed and the 53 kd species, which was purified on an SRE-7 nucleotide aff inity from HeLa cell nuclei (Wang et al., 1993) and whose peptide sequences were employed to design the oligonuleotide primers needed for the cloning of these cDNAs (Yokoyama et al., 1993). This size discrepancy and the cDNA sequence alerted the Brown and Goldstein laboratory to the possibility that these SREBPs were unorthodox transcription factors. Wang et al. (1994 [this issueof Ce//)) present compelling evidence that SREBP-1 is synthesized as a 125 kd high molecular mass precursor that is bound to the ER and the nuclear envelope as an integral membrane protein. In sterol-depleted HeLa cells, a significant fraction of this precursor is cleaved proteolytically to release from the membrane a 59 kd species that enters the nucleus to stimulate transcription (Figure 1). This conversion is easily observed in the presence of a neutral cysteine protease inhibitor that stabilizes the mature 39 kd species; the protein and its precursor otherwise turn over rapidly. Addition of sterol to the HeLa cells inhibits this conversion but does not appear to affect SREBP-7 mRNA levels, suggesting that the regulation of SREBP-1 is predominantlyposttranslational. Simian COS cells overexpressing a full-length or an amino-terminal form (residues 1410) of the hamster SREBP-1 demonstrated that the full-length form is in the extranuclear compartment. In contrast, the truncated form is entirely in the nucleus, indicating that this fragment (residues l-410) contains a nuclear localization signal. SREBP-1 is a transcription factor that is excluded from the nucleus specifically by its attachment to the ER membrane. Upon reduction in the concentration of cellular sterol, the cleaved precursor containing the basic-helixloop-helix-leucine zipper transcription factor domain is transported into the nucleus. A similar strategy of extranuclear confinement is also used in the case of the NF-KBIRel family of transcription factors (Liou and Baltimore, 1993); the inactive forms are maintained in the cytoplasm until multiple regulatory influences lead to their activation and transport to the nucleus. They all share a Rel homology domain region of about 300 amino acids that is responsible for DNA-binding site recognition, dimerization, nuclear localization, and interactions with agroupof nucleartranslocation inhibitory molecules, the IK-Bs. Two members of the NF-icB/Rel family arise from bipartite precursors; the amino termini contain the transcription factor moieties whereas the carboxyl termini have ankyrin repeats that may serve as integral regulators to mask the nuclear localization signal in the Rel homology domain. Whether the proteolytic cleavage of ~105 or ~100 NF-KBIRel precursors to produce ~50 and ~52, respectively, is a regulated process is still an open question. An inhibitory subunit, IK-8, forms a complex with the mature NF-KB subunit. Appropriate phosphorylation of IK-B causes it to dissociate from NF-KB, thereby leading to the activation and translocation of NF-KB. There are, however, several obvious differences between the regula-
#
Sterol inhibits cleavage
Decrease in sterol level allows cleavage
tion of the SREBP-1 and NF-~B/Ffel transcription factors. For example, whereas NF-KB/Rel is cytoplasmically sequestered, SREBP-1 is bound to the ER membrane. For NF-rcB/Rel, proteolytic cleavage does not appear to be a direct means for activation, while phosphorylation appears to be an important regulatory strategy. Although it is possible that phosphorylation of the aminoterminal portion of SREBP-1 (Wang et al., 1994) affects its activation, negative regulation of SREBP-1 cleavage by sterols plays a major regulatory role. An intriguing scenario is that phosphorylation of the SREBP-1 precursor bound to the membrane affects its proteolytic cleavage, as in the case of the colony-stimulating factor 1 (Downing et al., 1989) and interleukin-3 (Mui et al., 1993) receptors, thereby leading to increased activation or turnover of the SREBP-1 precursor. Alternatively, the observed phosphorylation may have consequences for nuclear binding of the active forms as home- or heterodimers. Why does the cell simply not use a liganddependent transcription factor like the steroid hormone and related receptors (Parker, 1993; Truss and Beato, 1993) to downregulate transcription of LDL receptor, HMG CoA synthase, and HMG CoA reductase? The steroid hormone and related receptors (retinoid, thyroid, vitamin D) have been shown to shuttle constantly between the nucleus and cytoplasm but appear to be predominantly nuclear upon ligand binding because they are actively transported into the nucleus. Upon hormone binding, these steroid hormone receptors bind to response elements near their target genes and stimulate (or, in some cases, inhibit) transcription. One reason for not using this strategy may be that the cell needs to sense cholesterol or a related sterol concentration in the membrane, more specifically in the ER, the intracellular site of sterol biogenesis, and relay this signal to modulate transcription in the nucleus. In eukaryotic cells, the accumulation of unfolded proteins in the ER triggers a signaling pathway from the ER to the nucleus. This pathway may involve a gene product similar to the yeast cdc2+/CDC28related transmembrane kinase
Figure 1. Cholesterol Regulates Cleavage of the Membrane-Bound SREBP-1 Precusor A decrease in ER membrane cholesterol allows proteolytic cleavage of the membrane-bound SREBP-1 precursor. The 68 kd cleavage product is transported into the nucleus by a nuclear pore complex, binds to SRE-1, and activates transcription of genes flanked by this element, such as the LDL receptor gene.
(Cox et al., 1993; Mori et al., 1993) which responds to a decrease in free BiP (chaperone) or an increase BiPunfolded protein complex with an increase in its serinel threonine kinase activity. This kinase sensor appears to activate directly a specific set of transcription factors such as unfolded protein response factor (known as UPRF), which binds the 22 bp element that is necessary and sufficient for the induction of BiP mRNA in response to unfolded proteins (Mori et al., 1993). SREBP-1, however, may combine the membrane bound sensor and transcrip tion factor functions in a single protein that responds to changes in ER membrane sterol concentrations. What is the function of membrane cholesterol and why is its regulation so crucial in the ER? Cholesterol and related sterols occur in the plasma membranes of eukaryotic cells in concentrations equimolar to that of all other lipids combined, whereas the ER, nuclear envelope, and mitochondria are poor in cholesterol (Urbani and Simoni, 1990; Lange, 1992; Bretscher and Munro, 1993). Sterols cannot be essential for membrane integrity because bacteria do not have them. The principal role of cholesterol in the plasma membrane may be to render this bilayer less permeable to small molecules (Bretscher and Munro, 1993). The multiple cis doubfe bonds in acyl chains of plasma membrane phospholipids decrease their packing efficiency, allowing them to remain in the liquid state at ambient temperatures but also permitting transient cavities to form and serve as channels for small water-soluble solutes to pass. Introduction of cholesterol has two principal effects on a lipid bffayer (Bretscher and Munro, 1993). Fatty acid side chains that abut the fused ring system of cholesterol become less deformable, and the acyl side chain methylene groups (2-10) become ordered and tightly packed, thereby reducing the bilayer permeability. Second, the cholesterol-ordered segments of the acyl side chains now lie perpendicular to the bifayer, thereby increasing its thickness by as much as 20% in a model phophatidylcholine phospholipid bilayer (Bretscher and Munro, 1993). Membrane proteins are inserted by translo-
$nireview
cation chaperones into eukaryotic cell membranes in the ER, a membrane that contains a fraction of the cholesterol of the plasma membrane, utilizing the transiently formed cavities in this cholesterol pcor lipid bilayer. In these cells, cholesterol is synthesized in the ER and removed by transport through the Golgi to reach the plasma membrane. This cholesterol gradient is necessary to decrease small solute permeability at the cholesterol-rich plasma membrane, to permit membrane protein assembfy in the cho lesterol-poor ER, and to provide a mechanism for the sorting of membrane proteins of the Golgi apparatus (Bretscher and Munro, 1993). Examination of the transmembrane domains of known Golgi proteins reveal that they contain two features that may promote their exclusion from cholesterol-rich membrane, such as of those vesicles bound for the plasma membrane. They contain consistently shorter transmembrane domain than that found in plasma membrane proteins (17 versus 22 residues) and bulky side chains, both of which are energetically unfavorable in the thicker and more tightly packed bilayer of the cholesterol rich membrane. The presence of a thicker cholesterol- and sphingomyelin-rich membrane bilayer beyond the Golgi may explain the retention of Golgi membrane proteins in a process that transports cholesterol and other membrane proteins to the plasma membrane (Bretscher and Munro, 1993). It is tempting to propose that the cholesterol-mediated change in the bilayer thickness may be the trigger for the cleavage of the SREBP-I precursor to the mature 65 kd species. The 125 kd SREBP-1 precursor that is bound to the ER membranes is an integral membrane protein. This precursor contains two hydrophobic stretches of 22 and 21 amino acids, respectively. Both or either one of these stretches could serve as a transmembrane segment. As the amino terminus of SREBP-1 is translocated to the nucleus, presumably through a nuclear pore complex, it must face the cytoplasm. The carboxyl terminus, however, can face the lumen of the ER or the cytoplasm. An increase in the sterol concentration could inhibit cleavage of the precursor by acting on the protease, by causing a conformational change in the membrane bound precursor, or by affecting the ER membrane itself. A small change in cholesterol content of the relatively cholesterol-poor ER membrane may cause the bilayer to expand by several angstroms, thereby shielding a proteolytic site on the SREBP-1 precursor. The unprocessed precursor would then turn over rather than undergo proteolytic conversion to the mature form. The low cholesterol content of the ER membrane makes a small change in free cholesterol far easier to detect than at the cholesterol-rich plasma membrane. The combination of a membrane-bound sensor (in this case, for cholesterol) and a transcription factor in a single molecule is an interesting and unique approach for relaying a signal from the ER membrane to the nucleus. Future studies will determine what mechanism is involved in the processing of SREBPs, whether phosphorylation of SREBP-1 plays a regulatory role, and how the aminoterminal portions oligomerize before binding to the specific DNA sequences. In addition, we also need to know
whether the 65 kd proteolytic fragment requires the assistance of other proteins (chaperones) in maintaining ftsoptimal conformation as it is transported from the cytoplasm to the nucleus. Finally, other examples of membranabound transcription factors that play important roles in intra- or intercellular signaling may also be found.
Bretscher, M. S.; and Munro, S. (1993). Sciince 267.1280-1291. Cm J. S., Shamu, C. E., and Walter, P. (1993). Cell 731197-1208. Downing. J. Ft., ROUSS~I,M. F.. and Sherr. C. J. (1999). M~I. cetl. Bid. 9, 29962999. Gil, G., Faust, J. R., Chin, D. J., Gddstein. J. L., and Brown, M. S. (1995). Cell 47, 249-299. Goldstein. J. L.. and Brown, M. S. (199tI). Nature 343. 42343g Hua,x., Yokoyama,C., Wu, J., Brigge,hf. R., &own, M. S., Goldstein, J. L., and Wang, X. (1993). Pros. Natt. Acad. Sci. USA 90, 119W11607. Lange, Y. (1992). J. Lipid Res. 33.315-321. Liou, H. C., and Sattimore, 0. (1993). Curr. Opin. Cell Sit. 5, 477497. Mori. K., Ma, W., Gething, M.J., and Sambrook, J. (1993). cell 74, 743-799. Mui, A. L.-F., Kay. R. J., Humphries. K., and Krystal, G. (1993). Pruc. Natl. Acad. Sci. USA 89, 10812-10816. Murre, C., and Saltknore, D. (1992). In Transcriptional Regulation, S. L. Knight and K. R. Yamamoto, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), pp. 991-979. Pabo, C. 0.. and Sauer, R. T. (1992). Annu. Rev. Bbchem. 61, lt7331095. Parker, M. G. (1993). Curr. Opin. Cell Siil. 5, 499-504. Truss, M., and Seato, M. (1993). Endocrtnot. Rev. 14,429-479. Urbani, L., and Simoni, R. D. (199@. J. Bid. Chern. 265. 1919-1923. Wang, X., Sriggs, M. R., Hua, X., Yokoyama, C., Gotdstein, J. L., and Brown, M. S. (1993). J. BitI. Chern. 289. 14497-14504. Wang, X., Sate, R., Brown, M. S., Hua, X., and Goldstein, J. L. (1994). Cell 77, this issue. Yokwama, C., Wang, X., Brlggs, M. R., Admon, A., Wu, J., Hua, X., Goldstein. J. L., and Brown, M. S. (1993). Cell 75, 187-197.
Basic-Helix-Loop-Helix Transcription Factor and Sterol Sensor in a Single Membrane-Bound Molecule Gregory P. Gaslc Cell Press 50 Church Street Cambridge, Massachusetts 02139
Cholesterol and closely related sterols are present in the membranes of most eukaryoticcells, although they are not uniformly distributed among the different cell membrane compartments. In vertebrates, most of the cholesterol synthesis takes place in the liver, where a small portion is incorporated into the membranes of the hepatocytes and the remainder is exported as bile acids or cholesterol esters. The bile acids, stored in the gallbladder as salts, are released in the small intestine to aid with lipid digestion. Cholesterol esters are carried by plasma lipoproteins to tissues to be stored or consumed. Animal cells can obtain their cholesterol endogenously by synthesis from acetylcoenzyme A (CoA) through mevalonate or exogenously by receptor-mediated endocytosis of plasma low density lipoprotein (LDL). These cells must balance the internal and external sources of cholesterol to sustain the synthesis of mevalonate, a important precursor for the isoprenoid building blocks that are required to make sterols, electron transport moieties, glycoproteins, and farnesylated proteins, while avoiding sterol over accumulation (Goldstein and Brown, 1990). This balance is achieved through feedback regulation of the LDL receptor and through at least two consecutive enzymes in mevalonate synthesis, 3-hydroxy-3-methylglutaryl CoA (HMG CoA) synthase and HMG CoA reductase. For HMG CoA reductase, sterols exercise both transcriptional control and posttranslational control (Gil et al., 19&S), promoting the increased degradation of the enzyme in the endoplasmic reticulum (ER). A 10 bp sequence in the 5’flanking region, designated sterol regulatory element 1 (SRE-l), controls LDL receptor transcription such that when cellular sterol stores are expended, the gene is transcribed, receptor biosynthesis increases, and LDL uptake through receptor-mediated endocytosis increases (Gold stein and Brown, 1990). Likewise, when cellular sterol stores are replenished, SRE-1 activity is abolished and LDL receptor transcription decreases. SRE-1 is a conditional positive element that enhances LDL receptor and HMG CoA synthase gene transcription in the absence but not presence of sterols. The cDNAs coding for two proteins, SRE-binding proteins 1 and 2 (SREBP-1 and SREBP-P), each of which binds SRE-1 with a nucleotide specificity that is in perfect agreement with that required for sterol-regulated transcription (Wang et al., 1993), have been characterized (Yokoyama et al., 1993; Hua et al., 1993). Both contain a sequence (amino acid residues 324-394 of SREBP-1) that conforms to the consensus sequence of the basic-helixloop-helix-leucine zipper family of transcription factors (Murre and Baltimore, 1992; Pabo and Sauer, 1992). HOWever, there is an interesting contrast between the 125 kd
Minireview
protein that is produced when these cDNAs are expressed and the 53 kd species, which was purified on an SRE-7 nucleotide aff inity from HeLa cell nuclei (Wang et al., 1993) and whose peptide sequences were employed to design the oligonuleotide primers needed for the cloning of these cDNAs (Yokoyama et al., 1993). This size discrepancy and the cDNA sequence alerted the Brown and Goldstein laboratory to the possibility that these SREBPs were unorthodox transcription factors. Wang et al. (1994 [this issueof Ce//)) present compelling evidence that SREBP-1 is synthesized as a 125 kd high molecular mass precursor that is bound to the ER and the nuclear envelope as an integral membrane protein. In sterol-depleted HeLa cells, a significant fraction of this precursor is cleaved proteolytically to release from the membrane a 59 kd species that enters the nucleus to stimulate transcription (Figure 1). This conversion is easily observed in the presence of a neutral cysteine protease inhibitor that stabilizes the mature 39 kd species; the protein and its precursor otherwise turn over rapidly. Addition of sterol to the HeLa cells inhibits this conversion but does not appear to affect SREBP-7 mRNA levels, suggesting that the regulation of SREBP-1 is predominantlyposttranslational. Simian COS cells overexpressing a full-length or an amino-terminal form (residues 1410) of the hamster SREBP-1 demonstrated that the full-length form is in the extranuclear compartment. In contrast, the truncated form is entirely in the nucleus, indicating that this fragment (residues l-410) contains a nuclear localization signal. SREBP-1 is a transcription factor that is excluded from the nucleus specifically by its attachment to the ER membrane. Upon reduction in the concentration of cellular sterol, the cleaved precursor containing the basic-helixloop-helix-leucine zipper transcription factor domain is transported into the nucleus. A similar strategy of extranuclear confinement is also used in the case of the NF-KBIRel family of transcription factors (Liou and Baltimore, 1993); the inactive forms are maintained in the cytoplasm until multiple regulatory influences lead to their activation and transport to the nucleus. They all share a Rel homology domain region of about 300 amino acids that is responsible for DNA-binding site recognition, dimerization, nuclear localization, and interactions with agroupof nucleartranslocation inhibitory molecules, the IK-Bs. Two members of the NF-icB/Rel family arise from bipartite precursors; the amino termini contain the transcription factor moieties whereas the carboxyl termini have ankyrin repeats that may serve as integral regulators to mask the nuclear localization signal in the Rel homology domain. Whether the proteolytic cleavage of ~105 or ~100 NF-KBIRel precursors to produce ~50 and ~52, respectively, is a regulated process is still an open question. An inhibitory subunit, IK-8, forms a complex with the mature NF-KB subunit. Appropriate phosphorylation of IK-B causes it to dissociate from NF-KB, thereby leading to the activation and translocation of NF-KB. There are, however, several obvious differences between the regula-
#
Sterol inhibits cleavage
Decrease in sterol level allows cleavage
tion of the SREBP-1 and NF-~B/Ffel transcription factors. For example, whereas NF-KB/Rel is cytoplasmically sequestered, SREBP-1 is bound to the ER membrane. For NF-rcB/Rel, proteolytic cleavage does not appear to be a direct means for activation, while phosphorylation appears to be an important regulatory strategy. Although it is possible that phosphorylation of the aminoterminal portion of SREBP-1 (Wang et al., 1994) affects its activation, negative regulation of SREBP-1 cleavage by sterols plays a major regulatory role. An intriguing scenario is that phosphorylation of the SREBP-1 precursor bound to the membrane affects its proteolytic cleavage, as in the case of the colony-stimulating factor 1 (Downing et al., 1989) and interleukin-3 (Mui et al., 1993) receptors, thereby leading to increased activation or turnover of the SREBP-1 precursor. Alternatively, the observed phosphorylation may have consequences for nuclear binding of the active forms as home- or heterodimers. Why does the cell simply not use a liganddependent transcription factor like the steroid hormone and related receptors (Parker, 1993; Truss and Beato, 1993) to downregulate transcription of LDL receptor, HMG CoA synthase, and HMG CoA reductase? The steroid hormone and related receptors (retinoid, thyroid, vitamin D) have been shown to shuttle constantly between the nucleus and cytoplasm but appear to be predominantly nuclear upon ligand binding because they are actively transported into the nucleus. Upon hormone binding, these steroid hormone receptors bind to response elements near their target genes and stimulate (or, in some cases, inhibit) transcription. One reason for not using this strategy may be that the cell needs to sense cholesterol or a related sterol concentration in the membrane, more specifically in the ER, the intracellular site of sterol biogenesis, and relay this signal to modulate transcription in the nucleus. In eukaryotic cells, the accumulation of unfolded proteins in the ER triggers a signaling pathway from the ER to the nucleus. This pathway may involve a gene product similar to the yeast cdc2+/CDC28related transmembrane kinase
Figure 1. Cholesterol Regulates Cleavage of the Membrane-Bound SREBP-1 Precusor A decrease in ER membrane cholesterol allows proteolytic cleavage of the membrane-bound SREBP-1 precursor. The 68 kd cleavage product is transported into the nucleus by a nuclear pore complex, binds to SRE-1, and activates transcription of genes flanked by this element, such as the LDL receptor gene.
(Cox et al., 1993; Mori et al., 1993) which responds to a decrease in free BiP (chaperone) or an increase BiPunfolded protein complex with an increase in its serinel threonine kinase activity. This kinase sensor appears to activate directly a specific set of transcription factors such as unfolded protein response factor (known as UPRF), which binds the 22 bp element that is necessary and sufficient for the induction of BiP mRNA in response to unfolded proteins (Mori et al., 1993). SREBP-1, however, may combine the membrane bound sensor and transcrip tion factor functions in a single protein that responds to changes in ER membrane sterol concentrations. What is the function of membrane cholesterol and why is its regulation so crucial in the ER? Cholesterol and related sterols occur in the plasma membranes of eukaryotic cells in concentrations equimolar to that of all other lipids combined, whereas the ER, nuclear envelope, and mitochondria are poor in cholesterol (Urbani and Simoni, 1990; Lange, 1992; Bretscher and Munro, 1993). Sterols cannot be essential for membrane integrity because bacteria do not have them. The principal role of cholesterol in the plasma membrane may be to render this bilayer less permeable to small molecules (Bretscher and Munro, 1993). The multiple cis doubfe bonds in acyl chains of plasma membrane phospholipids decrease their packing efficiency, allowing them to remain in the liquid state at ambient temperatures but also permitting transient cavities to form and serve as channels for small water-soluble solutes to pass. Introduction of cholesterol has two principal effects on a lipid bffayer (Bretscher and Munro, 1993). Fatty acid side chains that abut the fused ring system of cholesterol become less deformable, and the acyl side chain methylene groups (2-10) become ordered and tightly packed, thereby reducing the bilayer permeability. Second, the cholesterol-ordered segments of the acyl side chains now lie perpendicular to the bifayer, thereby increasing its thickness by as much as 20% in a model phophatidylcholine phospholipid bilayer (Bretscher and Munro, 1993). Membrane proteins are inserted by translo-
$nireview
cation chaperones into eukaryotic cell membranes in the ER, a membrane that contains a fraction of the cholesterol of the plasma membrane, utilizing the transiently formed cavities in this cholesterol pcor lipid bilayer. In these cells, cholesterol is synthesized in the ER and removed by transport through the Golgi to reach the plasma membrane. This cholesterol gradient is necessary to decrease small solute permeability at the cholesterol-rich plasma membrane, to permit membrane protein assembfy in the cho lesterol-poor ER, and to provide a mechanism for the sorting of membrane proteins of the Golgi apparatus (Bretscher and Munro, 1993). Examination of the transmembrane domains of known Golgi proteins reveal that they contain two features that may promote their exclusion from cholesterol-rich membrane, such as of those vesicles bound for the plasma membrane. They contain consistently shorter transmembrane domain than that found in plasma membrane proteins (17 versus 22 residues) and bulky side chains, both of which are energetically unfavorable in the thicker and more tightly packed bilayer of the cholesterol rich membrane. The presence of a thicker cholesterol- and sphingomyelin-rich membrane bilayer beyond the Golgi may explain the retention of Golgi membrane proteins in a process that transports cholesterol and other membrane proteins to the plasma membrane (Bretscher and Munro, 1993). It is tempting to propose that the cholesterol-mediated change in the bilayer thickness may be the trigger for the cleavage of the SREBP-I precursor to the mature 65 kd species. The 125 kd SREBP-1 precursor that is bound to the ER membranes is an integral membrane protein. This precursor contains two hydrophobic stretches of 22 and 21 amino acids, respectively. Both or either one of these stretches could serve as a transmembrane segment. As the amino terminus of SREBP-1 is translocated to the nucleus, presumably through a nuclear pore complex, it must face the cytoplasm. The carboxyl terminus, however, can face the lumen of the ER or the cytoplasm. An increase in the sterol concentration could inhibit cleavage of the precursor by acting on the protease, by causing a conformational change in the membrane bound precursor, or by affecting the ER membrane itself. A small change in cholesterol content of the relatively cholesterol-poor ER membrane may cause the bilayer to expand by several angstroms, thereby shielding a proteolytic site on the SREBP-1 precursor. The unprocessed precursor would then turn over rather than undergo proteolytic conversion to the mature form. The low cholesterol content of the ER membrane makes a small change in free cholesterol far easier to detect than at the cholesterol-rich plasma membrane. The combination of a membrane-bound sensor (in this case, for cholesterol) and a transcription factor in a single molecule is an interesting and unique approach for relaying a signal from the ER membrane to the nucleus. Future studies will determine what mechanism is involved in the processing of SREBPs, whether phosphorylation of SREBP-1 plays a regulatory role, and how the aminoterminal portions oligomerize before binding to the specific DNA sequences. In addition, we also need to know
whether the 65 kd proteolytic fragment requires the assistance of other proteins (chaperones) in maintaining ftsoptimal conformation as it is transported from the cytoplasm to the nucleus. Finally, other examples of membranabound transcription factors that play important roles in intra- or intercellular signaling may also be found.
Bretscher, M. S.; and Munro, S. (1993). Sciince 267.1280-1291. Cm J. S., Shamu, C. E., and Walter, P. (1993). Cell 731197-1208. Downing. J. Ft., ROUSS~I,M. F.. and Sherr. C. J. (1999). M~I. cetl. Bid. 9, 29962999. Gil, G., Faust, J. R., Chin, D. J., Gddstein. J. L., and Brown, M. S. (1995). Cell 47, 249-299. Goldstein. J. L.. and Brown, M. S. (199tI). Nature 343. 42343g Hua,x., Yokoyama,C., Wu, J., Brigge,hf. R., &own, M. S., Goldstein, J. L., and Wang, X. (1993). Pros. Natt. Acad. Sci. USA 90, 119W11607. Lange, Y. (1992). J. Lipid Res. 33.315-321. Liou, H. C., and Sattimore, 0. (1993). Curr. Opin. Cell Sit. 5, 477497. Mori. K., Ma, W., Gething, M.J., and Sambrook, J. (1993). cell 74, 743-799. Mui, A. L.-F., Kay. R. J., Humphries. K., and Krystal, G. (1993). Pruc. Natl. Acad. Sci. USA 89, 10812-10816. Murre, C., and Saltknore, D. (1992). In Transcriptional Regulation, S. L. Knight and K. R. Yamamoto, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), pp. 991-979. Pabo, C. 0.. and Sauer, R. T. (1992). Annu. Rev. Bbchem. 61, lt7331095. Parker, M. G. (1993). Curr. Opin. Cell Siil. 5, 499-504. Truss, M., and Seato, M. (1993). Endocrtnot. Rev. 14,429-479. Urbani, L., and Simoni, R. D. (199@. J. Bid. Chern. 265. 1919-1923. Wang, X., Sriggs, M. R., Hua, X., Yokoyama, C., Gotdstein, J. L., and Brown, M. S. (1993). J. BitI. Chern. 289. 14497-14504. Wang, X., Sate, R., Brown, M. S., Hua, X., and Goldstein, J. L. (1994). Cell 77, this issue. Yokwama, C., Wang, X., Brlggs, M. R., Admon, A., Wu, J., Hua, X., Goldstein. J. L., and Brown, M. S. (1993). Cell 75, 187-197.