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More than 'ARF a story
HEADLINES
c? Yr
A CDK involved in the initiation transcription A kinase-cyclin
LIAO, S-M. et al. (1995) pair in the RNA polymerase Nature 374, 193-l 96
of
II holoenzyme
Genetic suppression of C-terminal domain (CTD) mutations in RNA polymerase II has facilitated the identification of several of its regulatory proteins in the budding yeast Saccharomyces cerevisiae. These regulators have been termed suppressors of RNA polymerase B (SRB proteins). Here, Liao et al. characterize the latest members of the SRB family, SRBlO and SRBl7, mutant alleles of which specifically suppress the conditional phenotypes of CTD truncation mutations. SRBlO encodes a 541 amino acid protein kinase previously identified as UME.5, a regulator of meiosis-specific genes. SRBl O/UMfS shows considerable protein sequence identity to a number of yeast serine/threonine protein kinases, including CDC28. SRBl7 encodes a 323 amino acid protein that is 28% identical and 58% similar in sequence to both human and Drosophila cyclin C and contains a motif partially identical to the conserved ‘cyclin box’that is thought to be involved in the interaction between kinase and cyclin subunits. The authors present genetic and biochemical evidence that SRBI 0 and SRBI 1 form a kinase-cyclin pair in the RNA polymerase II holoenzyme. Moreover, their inclusion in this complex is essential for a normal transcriptional response to galactose induction in vivo. Point mutations in SRBlO that inactivate kinasefunction without affecting incorporation into the holoenzyme result in as much as a lOO-fold reduction in transcriptional induction. Furthermore, Liao et al. show that CTD phosphotylation in vitro is strongly dependent on wild-type SRBI 0. Although the exact role of CTD phosphorylation in the regulation of transcription remains to be determined, S. cerevisiae transcriptional responses clearly involve the SRB kinase-cyclin pair. = fr
More than Overexpression perturbations
‘ARF a story
PETERS, P. J. et al. (1995) of wild-type and mutant ARFl and ARF6: distinct of nonoverlapping membrane compartments /, Cell Biol. 128, 1003-I 017
SCHOREY, C. D., LI, C., COLOMBO, M. I. and STAHL, P. D. (1995) A regulatory role for ARF6 in receptor mediated endocytosis Science 267, 1175-l 178 It has recently become clear that small CTPases of the ADP-ribosylation factor (ARF) family are involved in the regulation of eukaryotic membrane transport. At least six ARF genes have been cloned. With the exception of ARFI, which is localized to the Golgi complex and required for assembly of the COP1 nonclathrin coatomer complex, little was known about the location and function of the other members of the ARFfamily. Stahl’s group previously documented that early endosome dynamics was regulated by an unknown ARF protein. Peters et al. and Schorey et al. now report that ARF6 is associated with the plasma membrane and with early endocytic structures that contain the transferrin receptor (TfR). Overexpression of ARF6 orARF6/Q67L, a mutant protein that does not hydrolyse CTP, led to their redistribution to the plasma membrane, and produced a significant redistribution of the TfR to the cell surface. In contrast, ARF6/T27N, a mutant protein that does not bind CTP, accumulated exclusively in intracellular vesicles together with the TfR, whose cell-surface expression was decreased by 70%. Interestingly, the tubulovesicular membranes in the cells expressing ARF6/T27N were surrounded by a coat of electron-dense material that was distinct from both COP1 and the clathrin coat. Although it is unclear which step of the endocytic route is regulated by ARM, the exciting implication of these two papers is that ARM might be involved in the assembly of a novel endosomal coat that is associated with early endocytic membranes. TRENDS IN CELL BIOLOGY VOL. 5 JUNE 1995
Rho-family proteins in fly development HARDEN, N., LOH, H. Y., CHIA, W. and LIM, L. (1995) A dominant inhibitory version of the small CTP-binding protein Rat disrupts cytoskeletal structures and inhibits developmental cell shape changes in Drosophila Development 121, 903-914 The Rho family of Ras-related proteins plays an important role in the organization of focal adhesion complexes and actin filaments, and includes Rat proteins, which induce plasma membrane ruffling and actin filament reorganization when microinjected into mammalian cells. As is the case for Ras proteins, dominant inhibitory Rat proteins can be constructed by mutating amino acid 17 from Thr to Asn. The dominant negative mutant probably acts by sequestering guanine nucleotide exchange factors that convert Rat to the active GTP-bound state. In this report, heat-shock-induced expression of dominant negative Rat (DRacA) in Drosophila embryos caused a number of developmental defects that may relate to cytoskeletal organization. Over 50% of the embryos showed germ-band retraction defects and had a hole or scab in the dorsal cuticle. The expression of dominant inhibitory DracA possibly causes a failure of the reorganization or contraction of the cytoskeleton, thus inhibiting closure of the dorsal cuticle. Immunofluorescence microscopy of actin and myosin in mutant and wildtype embryos confirmed the idea that Rat is involved in organizing these cytoskeletal proteins. Harden et al. observed a correlation between lack of accumulation of filamentous actin and myosin in epidermal cells and the severity of lack of dorsal closure. These defects are similar to those observed in zip mutants. In addition to dorsal closure, defects in head involution that resemble defects in car and zip embryos were observed. A lack of accumulation of a-spectrin, another membraneskeleton protein, was observed in epidermal cells near the dorsal opening of mutant DRacA embryos. Previous studies of cells in culture have shown that Rho-family members control dramatic cell-shape changes and movements. No doubt these proteins will be found to play essential roles in the cytoskeletal changes that underlie many aspects of development.
This month’s headlines were contributed by Donald Cullberg, David Hatton, Laura Machesky, Peter van der Sluijs, Carl Smythe, Dave Stuart, Debbie Sweet, Sylvie Tournier and Bill Wells.
221
c? Yr
A CDK involved in the initiation transcription A kinase-cyclin
LIAO, S-M. et al. (1995) pair in the RNA polymerase Nature 374, 193-l 96
of
II holoenzyme
Genetic suppression of C-terminal domain (CTD) mutations in RNA polymerase II has facilitated the identification of several of its regulatory proteins in the budding yeast Saccharomyces cerevisiae. These regulators have been termed suppressors of RNA polymerase B (SRB proteins). Here, Liao et al. characterize the latest members of the SRB family, SRBlO and SRBl7, mutant alleles of which specifically suppress the conditional phenotypes of CTD truncation mutations. SRBlO encodes a 541 amino acid protein kinase previously identified as UME.5, a regulator of meiosis-specific genes. SRBl O/UMfS shows considerable protein sequence identity to a number of yeast serine/threonine protein kinases, including CDC28. SRBl7 encodes a 323 amino acid protein that is 28% identical and 58% similar in sequence to both human and Drosophila cyclin C and contains a motif partially identical to the conserved ‘cyclin box’that is thought to be involved in the interaction between kinase and cyclin subunits. The authors present genetic and biochemical evidence that SRBI 0 and SRBI 1 form a kinase-cyclin pair in the RNA polymerase II holoenzyme. Moreover, their inclusion in this complex is essential for a normal transcriptional response to galactose induction in vivo. Point mutations in SRBlO that inactivate kinasefunction without affecting incorporation into the holoenzyme result in as much as a lOO-fold reduction in transcriptional induction. Furthermore, Liao et al. show that CTD phosphotylation in vitro is strongly dependent on wild-type SRBI 0. Although the exact role of CTD phosphorylation in the regulation of transcription remains to be determined, S. cerevisiae transcriptional responses clearly involve the SRB kinase-cyclin pair. = fr
More than Overexpression perturbations
‘ARF a story
PETERS, P. J. et al. (1995) of wild-type and mutant ARFl and ARF6: distinct of nonoverlapping membrane compartments /, Cell Biol. 128, 1003-I 017
SCHOREY, C. D., LI, C., COLOMBO, M. I. and STAHL, P. D. (1995) A regulatory role for ARF6 in receptor mediated endocytosis Science 267, 1175-l 178 It has recently become clear that small CTPases of the ADP-ribosylation factor (ARF) family are involved in the regulation of eukaryotic membrane transport. At least six ARF genes have been cloned. With the exception of ARFI, which is localized to the Golgi complex and required for assembly of the COP1 nonclathrin coatomer complex, little was known about the location and function of the other members of the ARFfamily. Stahl’s group previously documented that early endosome dynamics was regulated by an unknown ARF protein. Peters et al. and Schorey et al. now report that ARF6 is associated with the plasma membrane and with early endocytic structures that contain the transferrin receptor (TfR). Overexpression of ARF6 orARF6/Q67L, a mutant protein that does not hydrolyse CTP, led to their redistribution to the plasma membrane, and produced a significant redistribution of the TfR to the cell surface. In contrast, ARF6/T27N, a mutant protein that does not bind CTP, accumulated exclusively in intracellular vesicles together with the TfR, whose cell-surface expression was decreased by 70%. Interestingly, the tubulovesicular membranes in the cells expressing ARF6/T27N were surrounded by a coat of electron-dense material that was distinct from both COP1 and the clathrin coat. Although it is unclear which step of the endocytic route is regulated by ARM, the exciting implication of these two papers is that ARM might be involved in the assembly of a novel endosomal coat that is associated with early endocytic membranes. TRENDS IN CELL BIOLOGY VOL. 5 JUNE 1995
Rho-family proteins in fly development HARDEN, N., LOH, H. Y., CHIA, W. and LIM, L. (1995) A dominant inhibitory version of the small CTP-binding protein Rat disrupts cytoskeletal structures and inhibits developmental cell shape changes in Drosophila Development 121, 903-914 The Rho family of Ras-related proteins plays an important role in the organization of focal adhesion complexes and actin filaments, and includes Rat proteins, which induce plasma membrane ruffling and actin filament reorganization when microinjected into mammalian cells. As is the case for Ras proteins, dominant inhibitory Rat proteins can be constructed by mutating amino acid 17 from Thr to Asn. The dominant negative mutant probably acts by sequestering guanine nucleotide exchange factors that convert Rat to the active GTP-bound state. In this report, heat-shock-induced expression of dominant negative Rat (DRacA) in Drosophila embryos caused a number of developmental defects that may relate to cytoskeletal organization. Over 50% of the embryos showed germ-band retraction defects and had a hole or scab in the dorsal cuticle. The expression of dominant inhibitory DracA possibly causes a failure of the reorganization or contraction of the cytoskeleton, thus inhibiting closure of the dorsal cuticle. Immunofluorescence microscopy of actin and myosin in mutant and wildtype embryos confirmed the idea that Rat is involved in organizing these cytoskeletal proteins. Harden et al. observed a correlation between lack of accumulation of filamentous actin and myosin in epidermal cells and the severity of lack of dorsal closure. These defects are similar to those observed in zip mutants. In addition to dorsal closure, defects in head involution that resemble defects in car and zip embryos were observed. A lack of accumulation of a-spectrin, another membraneskeleton protein, was observed in epidermal cells near the dorsal opening of mutant DRacA embryos. Previous studies of cells in culture have shown that Rho-family members control dramatic cell-shape changes and movements. No doubt these proteins will be found to play essential roles in the cytoskeletal changes that underlie many aspects of development.
This month’s headlines were contributed by Donald Cullberg, David Hatton, Laura Machesky, Peter van der Sluijs, Carl Smythe, Dave Stuart, Debbie Sweet, Sylvie Tournier and Bill Wells.
221