Dominant-lethal α-tubulin mutants defective in microtubule depolymerization in yeast

被引:43
作者
Anders, KR [1 ]
Botstein, D [1 ]
机构
[1] Stanford Univ, Sch Med, Dept Genet, Stanford, CA 94305 USA
关键词
D O I
10.1091/mbc.12.12.3973
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
The dynamic instability of microtubules has long been understood to depend on the hydrolysis of GTP bound to beta -tubulin, an event stimulated by polymerization and necessary for depolymerization. Crystallographic studies of tubulin show that GTP is bound by beta -tubulin at the longitudinal dimer-dimer interface and contacts particular alpha -tubulin residues in the next dimer along the protofilament. This structural arrangement suggests that these contacts could account for assembly-stimulated GTP hydrolysis. As a test of this hypothesis, we examined, in yeast cells, the effect of mutating the alpha -tubulin residues predicted, on structural grounds, to be involved in GTPase activation. Mutation of these residues to alanine (i.e., D252A and E255A) created poisonous alpha -tubulins that caused lethality even as minor components of the alpha -tubulin pool. When the mutant alpha -tubulins were expressed from the galactose-inducible promoter of GAL1, cells rapidly acquired aberrant microtubule structures. Cytoplasmic microtubules were largely bundled, spindle assembly was inhibited, preexisting spindles failed to completely elongate, and occasional, stable microtubules were observed unattached to spindle pole bodies. Time-lapse microscopy showed that microtubule dynamics had ceased. Microtubules containing the mutant proteins did not depolymerize, even in the presence of nocodazole. These data support the view that alpha -tubulin is a GTPase-activating protein that acts, during microtubule polymerization, to stimulate GTP hydrolysis in beta -tubulin and thereby account for the dynamic instability of microtubules.
引用
收藏
页码:3973 / 3986
页数:14
相关论文
共 43 条
[1]   Structural transitions at microtubule ends correlate with their dynamic properties in Xenopus egg extracts [J].
Arnal, I ;
Karsenti, E ;
Hyman, AA .
JOURNAL OF CELL BIOLOGY, 2000, 149 (04) :767-774
[2]   BEHAVIOR OF SPINDLES AND SPINDLE PLAQUES IN CELL-CYCLE AND CONJUGATION OF SACCHAROMYCES-CEREVISIAE [J].
BYERS, B ;
GOETSCH, L .
JOURNAL OF BACTERIOLOGY, 1975, 124 (01) :511-523
[3]   DUPLICATION OF SPINDLE PLAQUES AND INTEGRATION OF YEAST-CELL CYCLE [J].
BYERS, B ;
GOETSCH, L .
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY, 1973, 38 :123-131
[4]   THE FREE-ENERGY FOR HYDROLYSIS OF A MICROTUBULE-BOUND NUCLEOTIDE TRIPHOSPHATE IS NEAR ZERO - ALL OF THE FREE-ENERGY FOR HYDROLYSIS IS STORED IN THE MICROTUBULE LATTICE [J].
CAPLOW, M ;
RUHLEN, RL ;
SHANKS, J .
JOURNAL OF CELL BIOLOGY, 1994, 127 (03) :779-788
[5]  
CAPLOW M, 1990, J BIOL CHEM, V265, P8935
[6]   Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex [J].
Carminati, JL ;
Stearns, T .
JOURNAL OF CELL BIOLOGY, 1997, 138 (03) :629-641
[7]   STRUCTURE OF GROWING MICROTUBULE ENDS - 2-DIMENSIONAL SHEETS CLOSE INTO TUBES AT VARIABLE RATES [J].
CHRETIEN, D ;
FULLER, SD ;
KARSENTI, E .
JOURNAL OF CELL BIOLOGY, 1995, 129 (05) :1311-1328
[8]   Novel roles for Saccharomyces cerevisiae mitotic spindle motors [J].
Cottingham, FR ;
Gheber, L ;
Miller, DL ;
Hoyt, MA .
JOURNAL OF CELL BIOLOGY, 1999, 147 (02) :335-349
[9]   MUTATIONS IN FTSZ THAT CONFER RESISTANCE TO SULA AFFECT THE INTERACTION OF FTSZ WITH GTP [J].
DAI, K ;
MUKHERJEE, A ;
XU, YF ;
LUTKENHAUS, J .
JOURNAL OF BACTERIOLOGY, 1994, 176 (01) :130-136
[10]   GUANOSINE-TRIPHOSPHATASE ACTIVITY OF TUBULIN ASSOCIATED WITH MICROTUBULE ASSEMBLY [J].
DAVIDPFEUTY, T ;
ERICKSON, HP ;
PANTALONI, D .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1977, 74 (12) :5372-5376