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Tick-dock
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of the mechanisms whereby lack of laminin a2 causes muscular dystrophy. In accordance with this, apoptosis was found in the muscles of homozygous dy mice. Earlier work from other groups with dystrophic mdx mice has shown that apoptosis precedes necrosis in the skeletal muscle of mdx mice. It will be a challenging task to compare extensively the mechanism of apoptosis induction in mdx mice and dy mice.
r Tick-dock CTPase
RYBIN, V. et al. (1996) activity of Rab5 acts as a timer endocytic membrane fusion Nature 383, 266-269
for
Protein and membrane transport occur by vesicle budding from donor membranes and subsequentvesicle docking and fusion at acceptor membranes. Regulation of these events ensures that proteins arrive at their proper destinations. Small CTP-binding proteins have been implicated in membrane traffic control, but the molecular mechanisms have not been elucidated. CTP hydrolysis by Rab proteins is postulated to be a component of the regulatory machinery. The current models for such regulation are that Rab CTP hydrolysis controls either membrane fusion, vesicle docking, or assembly of proteins on the transport vesicle prior to vesicle docking and fusion. To distinguish between these models, Rybin et al. engineered a Rab.S mutant, rab5D136N, that bound xanthosine 5’-diphosphate (XDP) with an affinity three orders of magnitude higher than it bound GDP. This mutant allowed the authors to study RabS-mediated transport events in isolation from other cellular GTPases. RabSD136N-XDP bound to earlyendosome-enriched membranes and incorporated and cytosoland ATPhydrolysed XTP. Rab5 D136N stimulated dependent early endosome fusion in a cell-free assay, and XDP-XTP exchange was required for fusion. These data show that rab5D136N functions in a manner analogous to wildtype Rab5. Further analysis showed that XTP hydrolysis occurred even when endosome fusion was blocked by omitting cytosol from the reaction. Conversely, addition of XTP$i to inhibit nucleotide hydrolysis, and of Rab GDP dissociation inhibitor to remove endogenous Rab5 from membranes, confirmed that membrane fusion and nucleotide triphosphate hydrolysis can be uncoupled. Since XTP hydrolysis and membrane fusion occur independently, Rab5 cannot be a molecular switch regulating fusion. Because the kinetics of XTP hydrolysis were independent of membrane concentration, it is unlikely that Rab proteins regulate vesicle docking. This leaves the third model: that nucleotide hydrolysis by Rab proteins regulates events prior to vesicle docking and fusion. The authors propose that Rab5 nucleotide exchange and hydrolysis regulate the membrane recruitment of rabaptin-5, a cytosolic protein that binds and stabilizes GTP-bound Rab5. In this model, the proportion of Rab5 with bound GTP, and the rate of GTP hydrolysis, determine the frequency of membrane fusion. Although the precise mechanism for Rab regulation of membrane fusion remains to be elucidated, the elegant experiments of Rybin and colleagues send us well on the way to a better understanding of the molecular mechanisms underlying membrane tiansport. trends
in CELL BIOLOGY
(Vol.
6) December
1996
mmw
fr Catching the bug for reversible phosphorylation YANG,
X., KANG, C. M., BRODY, M. S. and PRICE, C. W. (1996) Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress activate a bacterial transcription factor Genes Dev. 10,2265-2275
to
Regulation by reversible protein phosphorylation in bacteria is generally considered a minor phenomenon. This may be about to change-during their dissection of stress-response pathways in Bacillus subtilis, Yang et al. provide biochemical evidence for the reversible protein serine phosphorylation of two proteins and characterize the molecular interactions between the opposing protein serine kinases and phosphatases that catalyse these reactions. Their data may provide new insights into signal transduction by protein switching in eukaryotic cells. Energy-stress signals in B. subtilis are transduced by an interacting set of three proteins, RsbU, RsbV and RsbW, whereas environmental stress signals are mediated by these three proteins and three other proteins upstream of this cassette, namely RsbX, RsbS and RsbT. The target of these pathways is the transcription factor oB, which is unable to bind DNAwhen complexed with RsbW. Yang and colleagues show that each three-protein cassette consists of a protein serine kinase (RsbT and RsbW) and an opposing phosphatase (RsbX and RsbU). Reversible protein phosphorylation is shown to regulate protein-protein interactions between RsbT, RsbS and RsbU in both crosslinking and two-hybrid studies. In vivo, this is shown to regulate the release of cB from RsbW, and hence the transcription of genes required for the stress response. Also of novel interest is the finding that the protein serine kinase RsbT activates a protein serine phosphatase, RsbU, apparently in the absence of phosphotransfer to RsbU. If true, this represents a novel regulatory mechanism by protein switching, which may have broad implications - eukaryotic casein kinase II activity has already been shown to be regulated by ~34~~‘~ in the absence of phosphotransfer. Thus, these studies have two implications for eukaryotic signal transduction: the concern that proteins expressed in bacteria may, after all, be phosphorylated; and the possibility that protein kinases may be even more exciting than we thought.
(,(
.“‘,_
,,
:z:;j;;‘;;!. .):: .;;,;.;;j,:,;,: .‘,, I’. ‘_I ,) : “G,,,’
The head/&es section of &eqd; .in CEti?‘t$,’ :. BIOLOGY is intended to draw att&tiori a selection of research papers of impok:?’ tance to cell biology that have been published in the past few months. headfines are contributed regularly by a panel of research‘_ scientists appointed by the Editor. ” ;
459
of the mechanisms whereby lack of laminin a2 causes muscular dystrophy. In accordance with this, apoptosis was found in the muscles of homozygous dy mice. Earlier work from other groups with dystrophic mdx mice has shown that apoptosis precedes necrosis in the skeletal muscle of mdx mice. It will be a challenging task to compare extensively the mechanism of apoptosis induction in mdx mice and dy mice.
r Tick-dock CTPase
RYBIN, V. et al. (1996) activity of Rab5 acts as a timer endocytic membrane fusion Nature 383, 266-269
for
Protein and membrane transport occur by vesicle budding from donor membranes and subsequentvesicle docking and fusion at acceptor membranes. Regulation of these events ensures that proteins arrive at their proper destinations. Small CTP-binding proteins have been implicated in membrane traffic control, but the molecular mechanisms have not been elucidated. CTP hydrolysis by Rab proteins is postulated to be a component of the regulatory machinery. The current models for such regulation are that Rab CTP hydrolysis controls either membrane fusion, vesicle docking, or assembly of proteins on the transport vesicle prior to vesicle docking and fusion. To distinguish between these models, Rybin et al. engineered a Rab.S mutant, rab5D136N, that bound xanthosine 5’-diphosphate (XDP) with an affinity three orders of magnitude higher than it bound GDP. This mutant allowed the authors to study RabS-mediated transport events in isolation from other cellular GTPases. RabSD136N-XDP bound to earlyendosome-enriched membranes and incorporated and cytosoland ATPhydrolysed XTP. Rab5 D136N stimulated dependent early endosome fusion in a cell-free assay, and XDP-XTP exchange was required for fusion. These data show that rab5D136N functions in a manner analogous to wildtype Rab5. Further analysis showed that XTP hydrolysis occurred even when endosome fusion was blocked by omitting cytosol from the reaction. Conversely, addition of XTP$i to inhibit nucleotide hydrolysis, and of Rab GDP dissociation inhibitor to remove endogenous Rab5 from membranes, confirmed that membrane fusion and nucleotide triphosphate hydrolysis can be uncoupled. Since XTP hydrolysis and membrane fusion occur independently, Rab5 cannot be a molecular switch regulating fusion. Because the kinetics of XTP hydrolysis were independent of membrane concentration, it is unlikely that Rab proteins regulate vesicle docking. This leaves the third model: that nucleotide hydrolysis by Rab proteins regulates events prior to vesicle docking and fusion. The authors propose that Rab5 nucleotide exchange and hydrolysis regulate the membrane recruitment of rabaptin-5, a cytosolic protein that binds and stabilizes GTP-bound Rab5. In this model, the proportion of Rab5 with bound GTP, and the rate of GTP hydrolysis, determine the frequency of membrane fusion. Although the precise mechanism for Rab regulation of membrane fusion remains to be elucidated, the elegant experiments of Rybin and colleagues send us well on the way to a better understanding of the molecular mechanisms underlying membrane tiansport. trends
in CELL BIOLOGY
(Vol.
6) December
1996
mmw
fr Catching the bug for reversible phosphorylation YANG,
X., KANG, C. M., BRODY, M. S. and PRICE, C. W. (1996) Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress activate a bacterial transcription factor Genes Dev. 10,2265-2275
to
Regulation by reversible protein phosphorylation in bacteria is generally considered a minor phenomenon. This may be about to change-during their dissection of stress-response pathways in Bacillus subtilis, Yang et al. provide biochemical evidence for the reversible protein serine phosphorylation of two proteins and characterize the molecular interactions between the opposing protein serine kinases and phosphatases that catalyse these reactions. Their data may provide new insights into signal transduction by protein switching in eukaryotic cells. Energy-stress signals in B. subtilis are transduced by an interacting set of three proteins, RsbU, RsbV and RsbW, whereas environmental stress signals are mediated by these three proteins and three other proteins upstream of this cassette, namely RsbX, RsbS and RsbT. The target of these pathways is the transcription factor oB, which is unable to bind DNAwhen complexed with RsbW. Yang and colleagues show that each three-protein cassette consists of a protein serine kinase (RsbT and RsbW) and an opposing phosphatase (RsbX and RsbU). Reversible protein phosphorylation is shown to regulate protein-protein interactions between RsbT, RsbS and RsbU in both crosslinking and two-hybrid studies. In vivo, this is shown to regulate the release of cB from RsbW, and hence the transcription of genes required for the stress response. Also of novel interest is the finding that the protein serine kinase RsbT activates a protein serine phosphatase, RsbU, apparently in the absence of phosphotransfer to RsbU. If true, this represents a novel regulatory mechanism by protein switching, which may have broad implications - eukaryotic casein kinase II activity has already been shown to be regulated by ~34~~‘~ in the absence of phosphotransfer. Thus, these studies have two implications for eukaryotic signal transduction: the concern that proteins expressed in bacteria may, after all, be phosphorylated; and the possibility that protein kinases may be even more exciting than we thought.
(,(
.“‘,_
,,
:z:;j;;‘;;!. .):: .;;,;.;;j,:,;,: .‘,, I’. ‘_I ,) : “G,,,’
The head/&es section of &eqd; .in CEti?‘t$,’ :. BIOLOGY is intended to draw att&tiori a selection of research papers of impok:?’ tance to cell biology that have been published in the past few months. headfines are contributed regularly by a panel of research‘_ scientists appointed by the Editor. ” ;
459