Elevating the level of Cdc34/Ubc3 ubiquitin-conjugating enzyme in mitosis inhibits association of CENP-E with kinetochores and blocks the metaphase alignment of chromosomes.
Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.
Topper
L M
LM
Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.
Bastians
H
H
Ruderman
J V
JV
Gorbsky
G J
GJ
D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D002478:Cells, Cultured; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D002875:Chromosomes / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D003546:Cysteine Endopeptidases; D018386:Kinetochores / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008025:Ligases / Q000494:pharmacology*; D008677:Metaphase / Q000187:drug effects / Q000502:physiology*; D008845:Microinjections; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D008941:Spindle Apparatus; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases
D002868:Chromosomal Proteins, Non-Histone; D009097:Multienzyme Complexes; C068688:centromere protein E; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases