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Q: which came first? A: The Eg.
HEADLINES
Mutant mouse sheds light on storage-pool deficiency Storage-pool deficiencies are characterized by the inability of certain granules such as platelet dense granules, melanosomes and lysosomes to properly maintain their contents, resulting in defects in pigmentation, blood clotting and urinary secretion. Although a number of such mutations have been characterized in mice and humans, the mechanism underlying the common storage defects in these organelles is still poorly understood. Kantheti et al. have mapped one such mutation in the mocha mouse, a model for the Hermansky–Pudlak syndrome in humans. They find that the mocha phenotype is caused by a deletion in the gene encoding the d-subunit of the AP-3 adaptor-like complex, leading to premature truncation of AP-3 d and complete absence of the entire AP-3 complex1. The mutant mice, although viable, exhibit an unusual electroencephalogram,
are hyperactive and suffer from defects in blood clotting and reduced levels of renal lysosomal enzymes. Most importantly, in mocha brain, the synaptic-vesicle-associated ZnT-3 zinc transporter is reduced, resulting in a lack of zinc in presynaptic nerve terminals. These data suggest that AP-3 is responsible for cargo selection to lysosome-related organelles and might have an important role in the biogenesis of synaptic vesicles. But, how exactly are cargo proteins sorted into AP-3-coated vesicles? At least in the case of synaptic vesicle formation, v-SNAREs seem to hold the key. Salem et al. have investigated the effect of clostridial neurotoxins on the biogenesis of synaptic vesicles in vitro. They find that treatment of the donor membranes with tetanus toxin, which cleaves the v-SNARE VAMP/synaptobrevin 2, inhibits the formation and coating of synaptic
vesicles with AP-3 in a reconstituted PC12 cell system2. Hence, by serving to recruit AP-3 to the membrane, VAMP/synaptobrevin facilitates its own sorting into the newly formed vesicle. Thus, protein sorting and cargo selection during formation of coated vesicles appear to be important determinants in the biogenesis of storage organelles and might underlie the cause of an increasing number of granule-storage and neurological disorders. 1
2
Kantheti, P. et al. (1998) Mutation in AP-3 d in the mocha mouse links endosomal transport to storage deficiency in platelets, melanosomes, and synaptic vesicles, Neuron 21, 111–122 Salem, N. et al. (1998) A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex, Nat. Neurosci. 1, 551–556
Pole positioned by pombe pore protein To build a mitotic spindle, the yeast spindle pole body (SPB) must penetrate the nuclear envelope (NE), which never breaks down in yeast. Unlike the budding yeast Saccharomyces cerevisiae, whose SPB is always in the NE, the SPB of Schizosaccharomyces pombe is cytoplasmic during interphase and only enters the NE during mitosis. West et al. now implicate the cut11 gene in this process1. Cut11 mutants have a mitotic defect: the spindle is replaced by variable numbers of microtubule bundles, and chromosome segregation fails. The abnormal spindle arises from defective
SPB behaviour: instead of the normal pair of NE-embedded SPBs, serial sectioning reveals only one active SPB floating freely in the nucleoplasm, not anchored to the NE. Over time, SPBs accumulate in the cytoplasm, suggesting that SPBs duplicate normally but cannot insert into the NE. cut11 encodes a seven-transmembrane domain protein homologous to the S. cerevisiae NDC1 gene, which in budding yeast is needed to insert the nascent SPB into the NE. Cut11p localizes to nuclear pore complexes throughout the cell cycle and to the SPBs during mitosis. This mitosis-
specific localization is consistent with a role in SPB anchoring. Thus, although budding and fission yeast SPBs behave differently during the cell cycle, both appear to use a common machinery to insert themselves into the NE. 1
West, R. R., Vaisberg, E. V., Ding, R., Nurse, P. and McIntosh, J. R. (1998) cut111: a gene required for cell cycledependent spindle pole body anchoring in the nuclear envelope and bipolar spindle formation in Schizosaccharomyces pombe, Mol. Biol. Cell 9, 2839–2855
Q: which came first? A: The Eg. This month’s headlines were contributed by Volker Haucke, Chung Lau, Wallace Marshall, Robin May, Kirsten Sadler, Steven Theg and Peter Thomason
8
With the thousands of papers and perhaps an equal number of researchers working in the signalling field, it is a wonder that so little is known about the signalling pathway that leads from hormonal stimulation of oocytes to activation of the meiotic cell cycle. Progesterone is the mitogen for Xenopus oocytes, and, within minutes of progesterone stimulation, cAMP
levels and protein kinase A (PKA) activity drop and, several hours later, mos mRNA is translated, leading to activation of mitogen-activated protein (MAP) kinase. Within 6–8 hours of progesterone addition, maturationpromoting factor (MPF) is activated in a MAP-kinase-dependent manner, and the quiescent oocyte is catapulted into M phase. This paper provides the first
insight into what occurs during these long hours between the early and late progesterone-mediated events. Based on the presumption that signalling molecules are often modified when activated, and that their modification can be detected as a shift in mobility on SDS–PAGE, the authors designed a screen to identify oocyte proteins that ‘shift’ following
trends in CELL BIOLOGY (Vol. 9) January 1999
headlines progesterone stimulation1. They report the identification of two proteins that become modified following progesterone stimulation: one is unidentified and the other is the Eg2 protein. Endogenous Eg2 ‘upshifts’ on gels within 30 minutes of progesterone stimulation, presumably corresponding with its activation. Sequence analysis identifies Eg2 as a kinase, and recombinant Eg2 acquires kinase activity in vitro when mixed with extracts from progesterone-stimulated,
but not unstimulated, oocytes. Although overexpression of Eg2 does not trigger maturation on its own, it does accelerate the appearance of mos, activation of MAP kinase and germinal-vesicle breakdown. In addition, high Eg2 levels significantly reduce the amount of progesterone required to activate the cell cycle. Several immediate questions come to mind: what activates Eg2? What are the substrates of Eg2? Does Eg2 cause mos activation or stabilization of this
notoriously unstable protein? And what is this other protein (pTA10) that was identified by their screen? Perhaps this and subsequent studies will bring the much-neglected field of oocyte signalling up to speed with the rest of the signalling world.
proposed PtdIns(3)P 5-kinase1. Fab1p has a C-terminal phosphatidylinositol phosphate kinase domain, and an N-terminal RING FYVE domain, a type of zinc finger thought to bind specifically to PtdIns(3)P. Fab1p deletion strains fail to produce PtdIns(3,5)P2 and have elevated PtdIns(3)P. These cells have a grotesquely distended vacuole lacking proper acidification. Trafficking from the Golgi apparatus to the vacuole [which requires PtdIns(3)P] is intact, and so it appears that PtdIns(3,5)P2 is required at a later step, perhaps to recycle membrane to the Golgi or internalize it
into the vacuole. Fab1p seems to require at least one other protein, Vac7p, for activity, but sadly Vac7p has little homology to any known proteins. Working out its functions, as well as those of PtdIns(3,5)P2 itself, is sure to be under way soon.
1
Andrésson, T. and Ruderman, J. V. (1998) The kinase Eg2 is a component of the Xenopus oocyte progesteroneactivated signaling pathway, EMBO J. 17, 5627–5637
A fab result Fab1p is essential for the activity of PtdIns(3)P 5-kinase and the maintenance of vacuolar size and membrane homeostasis. The importance of inositol phospholipids, especially those phosphorylated at the D3 position of the inositol ring, is hard to overstate. We are becoming increasingly familiar with their roles in signal transduction and membrane traffic. Lately, a new member of the family, PtdIns(3,5)P2, has been welcomed. Emr and colleagues suggest that the source of this lipid in Saccharomyces cerevisiae is the phosphorylation of PtdIns(3)P by Fab1p, a
1
Gary, J. D., Wurmser, A. E., Bonangelino, C. J., Weisman, L. S. and Emr, S. D. (1998) Fab1p is essential for PtdIns(3)P 5-kinase activity and the maintenance of vacuolar size and membrane homeostasis, J. Cell Biol. 143, 65–79
Going green with every developmental stage Plastid biogenesis involves the import of most of the organelle proteins from the cytoplasm. While plastid homeostasis is ongoing during the lifetime of the organelle, it makes sense that the highest protein-import activity would be found in young, developing plastids during their biosynthesis. This notion was previously demonstrated experimentally1. However, the reasons underlying the high protein trafficking activity of young plastids were not known. The paper by Jarvis et al. provides perhaps the first glimpse into the mechanism of this developmental change in the efficiency of protein import into plastids. The authors isolated mutants in Arabidopsis that showed a pale green phenotype in young plants but recovered nearly wild-type appearance in older plants2. When they cloned the gene responsible for this
mutation, Toc33, they discovered a protein with a high degree of homology to Toc34, a polypeptide that is involved in protein import across the plastid envelope membranes. The mutant was shown to be deficient in its ability to import proteins into its chloroplasts, and this deficiency could be complemented by the Toc34 homologue. This demonstrates that there are two versions of the TOC34/33 protein, each with similar biological function. Interestingly, the newly discovered Toc33 is developmentally regulated, being fairly abundant in very young leaves and declining in abundance as the leaves mature. Together with the earlier observations of differential protein import efficiency in young and old leaves, this new finding suggests that the plastid import apparatus containing Toc33 may operate more efficiently than that containing Toc34.
trends in CELL BIOLOGY (Vol. 9) January 1999
As is often the case, this work raises as many questions as it answers. Why is there redundancy in this subunit of the import apparatus, and is there a similar redundancy in the other subunits? What properties of Toc33 improve protein translocation over that observed with Toc34? And why is Toc34, but not Toc33, crosslinked to incoming precursor proteins when the transcript level of Toc33 is always higher, even in mature tissue? The answers to these questions and others will clearly require more research – interestingly, in directions not anticipated just a few months ago. 1 2
Dahlin, C. and Cline, K. (1991) Plant Cell 3, 1131–1140 Jarvis, P., Chen, L-J., Li, H-M., Peto, C. A., Fankhauser, C. and Chory, J. (1998) An Arabidopsis mutant defective in the plastid general import apparatus, Science 282, 100–103
9
Mutant mouse sheds light on storage-pool deficiency Storage-pool deficiencies are characterized by the inability of certain granules such as platelet dense granules, melanosomes and lysosomes to properly maintain their contents, resulting in defects in pigmentation, blood clotting and urinary secretion. Although a number of such mutations have been characterized in mice and humans, the mechanism underlying the common storage defects in these organelles is still poorly understood. Kantheti et al. have mapped one such mutation in the mocha mouse, a model for the Hermansky–Pudlak syndrome in humans. They find that the mocha phenotype is caused by a deletion in the gene encoding the d-subunit of the AP-3 adaptor-like complex, leading to premature truncation of AP-3 d and complete absence of the entire AP-3 complex1. The mutant mice, although viable, exhibit an unusual electroencephalogram,
are hyperactive and suffer from defects in blood clotting and reduced levels of renal lysosomal enzymes. Most importantly, in mocha brain, the synaptic-vesicle-associated ZnT-3 zinc transporter is reduced, resulting in a lack of zinc in presynaptic nerve terminals. These data suggest that AP-3 is responsible for cargo selection to lysosome-related organelles and might have an important role in the biogenesis of synaptic vesicles. But, how exactly are cargo proteins sorted into AP-3-coated vesicles? At least in the case of synaptic vesicle formation, v-SNAREs seem to hold the key. Salem et al. have investigated the effect of clostridial neurotoxins on the biogenesis of synaptic vesicles in vitro. They find that treatment of the donor membranes with tetanus toxin, which cleaves the v-SNARE VAMP/synaptobrevin 2, inhibits the formation and coating of synaptic
vesicles with AP-3 in a reconstituted PC12 cell system2. Hence, by serving to recruit AP-3 to the membrane, VAMP/synaptobrevin facilitates its own sorting into the newly formed vesicle. Thus, protein sorting and cargo selection during formation of coated vesicles appear to be important determinants in the biogenesis of storage organelles and might underlie the cause of an increasing number of granule-storage and neurological disorders. 1
2
Kantheti, P. et al. (1998) Mutation in AP-3 d in the mocha mouse links endosomal transport to storage deficiency in platelets, melanosomes, and synaptic vesicles, Neuron 21, 111–122 Salem, N. et al. (1998) A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex, Nat. Neurosci. 1, 551–556
Pole positioned by pombe pore protein To build a mitotic spindle, the yeast spindle pole body (SPB) must penetrate the nuclear envelope (NE), which never breaks down in yeast. Unlike the budding yeast Saccharomyces cerevisiae, whose SPB is always in the NE, the SPB of Schizosaccharomyces pombe is cytoplasmic during interphase and only enters the NE during mitosis. West et al. now implicate the cut11 gene in this process1. Cut11 mutants have a mitotic defect: the spindle is replaced by variable numbers of microtubule bundles, and chromosome segregation fails. The abnormal spindle arises from defective
SPB behaviour: instead of the normal pair of NE-embedded SPBs, serial sectioning reveals only one active SPB floating freely in the nucleoplasm, not anchored to the NE. Over time, SPBs accumulate in the cytoplasm, suggesting that SPBs duplicate normally but cannot insert into the NE. cut11 encodes a seven-transmembrane domain protein homologous to the S. cerevisiae NDC1 gene, which in budding yeast is needed to insert the nascent SPB into the NE. Cut11p localizes to nuclear pore complexes throughout the cell cycle and to the SPBs during mitosis. This mitosis-
specific localization is consistent with a role in SPB anchoring. Thus, although budding and fission yeast SPBs behave differently during the cell cycle, both appear to use a common machinery to insert themselves into the NE. 1
West, R. R., Vaisberg, E. V., Ding, R., Nurse, P. and McIntosh, J. R. (1998) cut111: a gene required for cell cycledependent spindle pole body anchoring in the nuclear envelope and bipolar spindle formation in Schizosaccharomyces pombe, Mol. Biol. Cell 9, 2839–2855
Q: which came first? A: The Eg. This month’s headlines were contributed by Volker Haucke, Chung Lau, Wallace Marshall, Robin May, Kirsten Sadler, Steven Theg and Peter Thomason
8
With the thousands of papers and perhaps an equal number of researchers working in the signalling field, it is a wonder that so little is known about the signalling pathway that leads from hormonal stimulation of oocytes to activation of the meiotic cell cycle. Progesterone is the mitogen for Xenopus oocytes, and, within minutes of progesterone stimulation, cAMP
levels and protein kinase A (PKA) activity drop and, several hours later, mos mRNA is translated, leading to activation of mitogen-activated protein (MAP) kinase. Within 6–8 hours of progesterone addition, maturationpromoting factor (MPF) is activated in a MAP-kinase-dependent manner, and the quiescent oocyte is catapulted into M phase. This paper provides the first
insight into what occurs during these long hours between the early and late progesterone-mediated events. Based on the presumption that signalling molecules are often modified when activated, and that their modification can be detected as a shift in mobility on SDS–PAGE, the authors designed a screen to identify oocyte proteins that ‘shift’ following
trends in CELL BIOLOGY (Vol. 9) January 1999
headlines progesterone stimulation1. They report the identification of two proteins that become modified following progesterone stimulation: one is unidentified and the other is the Eg2 protein. Endogenous Eg2 ‘upshifts’ on gels within 30 minutes of progesterone stimulation, presumably corresponding with its activation. Sequence analysis identifies Eg2 as a kinase, and recombinant Eg2 acquires kinase activity in vitro when mixed with extracts from progesterone-stimulated,
but not unstimulated, oocytes. Although overexpression of Eg2 does not trigger maturation on its own, it does accelerate the appearance of mos, activation of MAP kinase and germinal-vesicle breakdown. In addition, high Eg2 levels significantly reduce the amount of progesterone required to activate the cell cycle. Several immediate questions come to mind: what activates Eg2? What are the substrates of Eg2? Does Eg2 cause mos activation or stabilization of this
notoriously unstable protein? And what is this other protein (pTA10) that was identified by their screen? Perhaps this and subsequent studies will bring the much-neglected field of oocyte signalling up to speed with the rest of the signalling world.
proposed PtdIns(3)P 5-kinase1. Fab1p has a C-terminal phosphatidylinositol phosphate kinase domain, and an N-terminal RING FYVE domain, a type of zinc finger thought to bind specifically to PtdIns(3)P. Fab1p deletion strains fail to produce PtdIns(3,5)P2 and have elevated PtdIns(3)P. These cells have a grotesquely distended vacuole lacking proper acidification. Trafficking from the Golgi apparatus to the vacuole [which requires PtdIns(3)P] is intact, and so it appears that PtdIns(3,5)P2 is required at a later step, perhaps to recycle membrane to the Golgi or internalize it
into the vacuole. Fab1p seems to require at least one other protein, Vac7p, for activity, but sadly Vac7p has little homology to any known proteins. Working out its functions, as well as those of PtdIns(3,5)P2 itself, is sure to be under way soon.
1
Andrésson, T. and Ruderman, J. V. (1998) The kinase Eg2 is a component of the Xenopus oocyte progesteroneactivated signaling pathway, EMBO J. 17, 5627–5637
A fab result Fab1p is essential for the activity of PtdIns(3)P 5-kinase and the maintenance of vacuolar size and membrane homeostasis. The importance of inositol phospholipids, especially those phosphorylated at the D3 position of the inositol ring, is hard to overstate. We are becoming increasingly familiar with their roles in signal transduction and membrane traffic. Lately, a new member of the family, PtdIns(3,5)P2, has been welcomed. Emr and colleagues suggest that the source of this lipid in Saccharomyces cerevisiae is the phosphorylation of PtdIns(3)P by Fab1p, a
1
Gary, J. D., Wurmser, A. E., Bonangelino, C. J., Weisman, L. S. and Emr, S. D. (1998) Fab1p is essential for PtdIns(3)P 5-kinase activity and the maintenance of vacuolar size and membrane homeostasis, J. Cell Biol. 143, 65–79
Going green with every developmental stage Plastid biogenesis involves the import of most of the organelle proteins from the cytoplasm. While plastid homeostasis is ongoing during the lifetime of the organelle, it makes sense that the highest protein-import activity would be found in young, developing plastids during their biosynthesis. This notion was previously demonstrated experimentally1. However, the reasons underlying the high protein trafficking activity of young plastids were not known. The paper by Jarvis et al. provides perhaps the first glimpse into the mechanism of this developmental change in the efficiency of protein import into plastids. The authors isolated mutants in Arabidopsis that showed a pale green phenotype in young plants but recovered nearly wild-type appearance in older plants2. When they cloned the gene responsible for this
mutation, Toc33, they discovered a protein with a high degree of homology to Toc34, a polypeptide that is involved in protein import across the plastid envelope membranes. The mutant was shown to be deficient in its ability to import proteins into its chloroplasts, and this deficiency could be complemented by the Toc34 homologue. This demonstrates that there are two versions of the TOC34/33 protein, each with similar biological function. Interestingly, the newly discovered Toc33 is developmentally regulated, being fairly abundant in very young leaves and declining in abundance as the leaves mature. Together with the earlier observations of differential protein import efficiency in young and old leaves, this new finding suggests that the plastid import apparatus containing Toc33 may operate more efficiently than that containing Toc34.
trends in CELL BIOLOGY (Vol. 9) January 1999
As is often the case, this work raises as many questions as it answers. Why is there redundancy in this subunit of the import apparatus, and is there a similar redundancy in the other subunits? What properties of Toc33 improve protein translocation over that observed with Toc34? And why is Toc34, but not Toc33, crosslinked to incoming precursor proteins when the transcript level of Toc33 is always higher, even in mature tissue? The answers to these questions and others will clearly require more research – interestingly, in directions not anticipated just a few months ago. 1 2
Dahlin, C. and Cline, K. (1991) Plant Cell 3, 1131–1140 Jarvis, P., Chen, L-J., Li, H-M., Peto, C. A., Fankhauser, C. and Chory, J. (1998) An Arabidopsis mutant defective in the plastid general import apparatus, Science 282, 100–103
9