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Centrioles get a helping hand from centrin
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News & Comment
TRENDS in Cell Biology Vol.12 No.11 November 2002
Cholesterol homeostasis: another twist on eSCAPe from the ER It is essential for mammalian cells to tightly regulate cholesterol content in their membranes. To this aim, they use a variety of feedback mechanisms that control cholesterol biosynthesis, storage as esters, and import from and export to circulating lipoproteins. The control of cellular cholesterol synthesis involves a fascinating series of regulated membrane-trafficking events. Recently, more details of the underlying mechanism have emerged. Key to cholesterol regulation is a family of membrane-bound transcription factors – sterol response element binding proteins (SREBPs) – which control enzyme levels in cholesterol synthesis, such as HMG-CoA reductase, as well as the synthesis of low-density lipoprotein (LDL)-receptors, which mediate endocytosis of cholesterol-rich LDL particles. Newly synthesized SREBPs form a complex in the endoplasmic reticulum (ER) with the polytopic membrane protein SREBP-cleavage activating protein (SCAP). To be activated, SREBPs must exit the ER complexed with their escort protein SCAP, and move to the Golgi. Here, SREBP is cleaved by two Golgi-localized proteases, S1P and S2P. This cleavage event releases
the soluble NH2-terminal domain of SREBP, which then can enter the nucleus as an active, positive transcription factor. Thus, SCAP plays a crucial role in the regulation of ER export of SREBPs via COP II coated vesicles. SCAP contains a sterol-sensing domain, a segment that includes five of its eight transmembrane helices. In situations of lowered cellular cholesterol, or when point mutations are introduced into the sterol sensing domain of SCAP, SCAP can exit the ER and escort SREBP to the Golgi complex. Now, Brown et al. demonstrate in vitro that cholesterol causes a conformational change in SCAP, as detected by the unmasking of closely spaced trypsin cleavage sites [1]. In addition, Yang et al. show that high cholesterol causes SCAP to bind to a novel ER membrane protein, INSIG-1, and that this strongly correlates with the inhibition of ER exit of SCAP, and SREBPs [2]. In sterol-depleted cells SCAP does not bind INSIG-1, thus enabling the SREBP/SCAP complex to leave the ER for the Golgi. Mutant SCAP that is unable to bind sterols also does not bind INSIG-1. Earlier studies from the Goldstein and Brown laboratory identified INSIG-1 as an mRNA with increased abundance in livers of transgenic
mice that overexpress SREBP-1a. The new data show that INSIG-1 acts in the regulated ER retention of SCAP. Finally, Espenshade et al. demonstrate that sterols block incorporation of SCAP into classic COP II-coated vesicles [3]. Furthermore, sterols block the Sar1p by dependent binding of the COP II proteins Sec23/34p to SCAP in vitro. These studies deepen our understanding of how ER exit of SCAP is controlled. As time goes by, we can be sure to expect more surprising insights into this sophisticated cellular feedback mechanism. 1 Brown, A. et al. (2002) Cholesterol addition to ER membranes alters conformation of SCAP, the SREBP escort protein that regulates cholesterol metabolism. Mol. Cell 10, 237–245 2 Yang, T. et al. (2002) Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110, 489–500 3 Espenshade, P. et al. (2002) Sterols block binding of COP II proteins to SCAP, thereby controlling SCAP sorting in ER. Proc. Natl. Acad. Sci. U. S. A. 99, 11694–11699
Rainer Duden [email protected]
Centrioles get a helping hand from centrin Although the precision of centriole duplication has aroused interest for over a century, its molecular mechanism remains almost entirely unknown. What proteins are required for a pre-existing centriole to induce assembly of a new centriole? One strong candidate is centrin, an EF hand-containing calcium binding protein first identified biochemically in green algae. Analysis of centrin mutants in the haploid unicellular alga Chlamydomonas (also known as ‘green yeast’ because of its powerful genetics) suggests that centrin is required for proper centriole segregation, and that defects in centrin lead to defects in centriole duplication. However, these studies leave several important questions about centrin function unanswered. First, centriole duplication still occurs in the Chlamydomonas centrin mutant, albeit to a reduced extent. Is this because the mutation, which was not a null, retains some residual centrin function, or because centrin is not strictly required for centriole duplication? http://tcb.trends.com
Second, although the centrioles of Chlamydomonas are virtually identical to those of animal cells, is it possible that centrin might play a different role in animals? To resolve these questions, Jeffrey Salisbury (who, by the way, was the first to discover centrin in algae) and colleagues have used RNA interference (RNAi) to reduce the levels of centrin-2 (the centriole-specific centrin isoform) in human tissue culture cells. RNAi of centrin-2 in HeLa cells leads to progressive loss of centrioles, consistent with a complete block in centriole replication. This result confirms that centrin is involved in centriole duplication in animal cells, which was suggested in previous work with algae. Moreover, these results show that a significant reduction in centrin levels can, in fact, cause a complete block in centriole duplication. This implies that the requirement for centrin is absolute. Interestingly, although centriole duplication is blocked, cell division continues
and bipolar spindles are still observed, even when centrioles are absent. However, these acentriolar spindles are abnormal, with broad, non-focused poles. In addition, multinucleate cells are observed. These results are consistent with a function for centrioles in both spindle assembly and cytokinesis. These results indicate that centrin plays a conserved role in centriole duplication in eukaryotes ranging from algae to animals. The yeast homolog of centrin, CDC31, is a component of the half-bridge structure involved in spindle-pole body duplication. Thus, these results suggest that centriole duplication might involve a structure similar to the half-bridge in yeast. 1 Salisbury, J.L. et al. (2002) Centrin-2 is required for centriole duplication in mammalian cells. Curr. Biol. 12, 1287–1292
Wallace Marshall [email protected]
0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
News & Comment
TRENDS in Cell Biology Vol.12 No.11 November 2002
Cholesterol homeostasis: another twist on eSCAPe from the ER It is essential for mammalian cells to tightly regulate cholesterol content in their membranes. To this aim, they use a variety of feedback mechanisms that control cholesterol biosynthesis, storage as esters, and import from and export to circulating lipoproteins. The control of cellular cholesterol synthesis involves a fascinating series of regulated membrane-trafficking events. Recently, more details of the underlying mechanism have emerged. Key to cholesterol regulation is a family of membrane-bound transcription factors – sterol response element binding proteins (SREBPs) – which control enzyme levels in cholesterol synthesis, such as HMG-CoA reductase, as well as the synthesis of low-density lipoprotein (LDL)-receptors, which mediate endocytosis of cholesterol-rich LDL particles. Newly synthesized SREBPs form a complex in the endoplasmic reticulum (ER) with the polytopic membrane protein SREBP-cleavage activating protein (SCAP). To be activated, SREBPs must exit the ER complexed with their escort protein SCAP, and move to the Golgi. Here, SREBP is cleaved by two Golgi-localized proteases, S1P and S2P. This cleavage event releases
the soluble NH2-terminal domain of SREBP, which then can enter the nucleus as an active, positive transcription factor. Thus, SCAP plays a crucial role in the regulation of ER export of SREBPs via COP II coated vesicles. SCAP contains a sterol-sensing domain, a segment that includes five of its eight transmembrane helices. In situations of lowered cellular cholesterol, or when point mutations are introduced into the sterol sensing domain of SCAP, SCAP can exit the ER and escort SREBP to the Golgi complex. Now, Brown et al. demonstrate in vitro that cholesterol causes a conformational change in SCAP, as detected by the unmasking of closely spaced trypsin cleavage sites [1]. In addition, Yang et al. show that high cholesterol causes SCAP to bind to a novel ER membrane protein, INSIG-1, and that this strongly correlates with the inhibition of ER exit of SCAP, and SREBPs [2]. In sterol-depleted cells SCAP does not bind INSIG-1, thus enabling the SREBP/SCAP complex to leave the ER for the Golgi. Mutant SCAP that is unable to bind sterols also does not bind INSIG-1. Earlier studies from the Goldstein and Brown laboratory identified INSIG-1 as an mRNA with increased abundance in livers of transgenic
mice that overexpress SREBP-1a. The new data show that INSIG-1 acts in the regulated ER retention of SCAP. Finally, Espenshade et al. demonstrate that sterols block incorporation of SCAP into classic COP II-coated vesicles [3]. Furthermore, sterols block the Sar1p by dependent binding of the COP II proteins Sec23/34p to SCAP in vitro. These studies deepen our understanding of how ER exit of SCAP is controlled. As time goes by, we can be sure to expect more surprising insights into this sophisticated cellular feedback mechanism. 1 Brown, A. et al. (2002) Cholesterol addition to ER membranes alters conformation of SCAP, the SREBP escort protein that regulates cholesterol metabolism. Mol. Cell 10, 237–245 2 Yang, T. et al. (2002) Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110, 489–500 3 Espenshade, P. et al. (2002) Sterols block binding of COP II proteins to SCAP, thereby controlling SCAP sorting in ER. Proc. Natl. Acad. Sci. U. S. A. 99, 11694–11699
Rainer Duden [email protected]
Centrioles get a helping hand from centrin Although the precision of centriole duplication has aroused interest for over a century, its molecular mechanism remains almost entirely unknown. What proteins are required for a pre-existing centriole to induce assembly of a new centriole? One strong candidate is centrin, an EF hand-containing calcium binding protein first identified biochemically in green algae. Analysis of centrin mutants in the haploid unicellular alga Chlamydomonas (also known as ‘green yeast’ because of its powerful genetics) suggests that centrin is required for proper centriole segregation, and that defects in centrin lead to defects in centriole duplication. However, these studies leave several important questions about centrin function unanswered. First, centriole duplication still occurs in the Chlamydomonas centrin mutant, albeit to a reduced extent. Is this because the mutation, which was not a null, retains some residual centrin function, or because centrin is not strictly required for centriole duplication? http://tcb.trends.com
Second, although the centrioles of Chlamydomonas are virtually identical to those of animal cells, is it possible that centrin might play a different role in animals? To resolve these questions, Jeffrey Salisbury (who, by the way, was the first to discover centrin in algae) and colleagues have used RNA interference (RNAi) to reduce the levels of centrin-2 (the centriole-specific centrin isoform) in human tissue culture cells. RNAi of centrin-2 in HeLa cells leads to progressive loss of centrioles, consistent with a complete block in centriole replication. This result confirms that centrin is involved in centriole duplication in animal cells, which was suggested in previous work with algae. Moreover, these results show that a significant reduction in centrin levels can, in fact, cause a complete block in centriole duplication. This implies that the requirement for centrin is absolute. Interestingly, although centriole duplication is blocked, cell division continues
and bipolar spindles are still observed, even when centrioles are absent. However, these acentriolar spindles are abnormal, with broad, non-focused poles. In addition, multinucleate cells are observed. These results are consistent with a function for centrioles in both spindle assembly and cytokinesis. These results indicate that centrin plays a conserved role in centriole duplication in eukaryotes ranging from algae to animals. The yeast homolog of centrin, CDC31, is a component of the half-bridge structure involved in spindle-pole body duplication. Thus, these results suggest that centriole duplication might involve a structure similar to the half-bridge in yeast. 1 Salisbury, J.L. et al. (2002) Centrin-2 is required for centriole duplication in mammalian cells. Curr. Biol. 12, 1287–1292
Wallace Marshall [email protected]
0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.