Sgt1 is required for human kinetochore assembly.
Budding yeast Sgt1 is required for kinetochore assembly, and its homologues have a role in cAMP signalling in fungi and pathogen resistance in plants. The function of mammalian Sgt1 is unknown. We report that RNA interference-mediated depletion of Sgt1 from HeLa cells causes dramatic alterations of the mitotic spindle and problems in chromosome alignment. Cells lacking Sgt1 undergo a mitotic delay due to activation of the spindle checkpoint. The checkpoint response, however, is significantly weakened in Sgt1-depleted cells, and this correlates with a dramatic reduction in kinetochore levels of Mad1, Mad2 and BubR1. These effects are explained by a problem in kinetochore assembly that prevents the localization of Hec1, CENP-E, CENP-F, CENP-I, but not CENP-C, to mitotic kinetochores. Our studies implicate Sgt1 as an essential protein and a critical assembly factor for the mammalian kinetochore, and lend credit to the hypothesis of a kinetochore assembly pathway that is conserved from yeast to man.
Steensgaard
Peter
P
Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy.
Garrè
Massimiliano
M
Muradore
Ivan
I
Transidico
Pietro
P
Nigg
Erich A
EA
Kitagawa
Katsumi
K
Earnshaw
William C
WC
Faretta
Mario
M
Musacchio
Andrea
A
D002135:Calcium-Binding Proteins / Q000032:analysis / Q000276:immunology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology*; D002503:Centromere / Q000276:immunology / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis / Q000276:immunology; D004268:DNA-Binding Proteins / Q000032:analysis / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism / Q000648:ultrastructure*; D064246:Mad2 Proteins; D008938:Mitosis / Q000235:genetics / Q000502:physiology; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000032:analysis / Q000276:immunology; D011494:Protein Kinases / Q000032:analysis / Q000276:immunology; D017346:Protein Serine-Threonine Kinases; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012097:Repressor Proteins / Q000032:analysis / Q000276:immunology; D008941:Spindle Apparatus / Q000235:genetics* / Q000378:metabolism
C484297:CENPI protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C501384:MAD1L1 protein, human; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; C490164:SUGT1 protein, human; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases