Pre-M phase-promoting factor associates with annulate lamellae in Xenopus oocytes and egg extracts.
We have used complementary biochemical and in vivo approaches to study the compartmentalization of M phase-promoting factor (MPF) in prophase Xenopus eggs and oocytes. We first examined the distribution of MPF (Cdc2/CyclinB2) and membranous organelles in high-speed extracts of Xenopus eggs made during mitotic prophase. These extracts were found to lack mitochondria, Golgi membranes, and most endoplasmic reticulum (ER) but to contain the bulk of the pre-MPF pool. This pre-MPF could be pelleted by further centrifugation along with components necessary to activate it. On activation, Cdc2/CyclinB2 moved into the soluble fraction. Electron microscopy and Western blot analysis showed that the pre-MPF pellet contained a specific ER subdomain comprising "annulate lamellae" (AL): stacked ER membranes highly enriched in nuclear pores. Colocalization of pre-MPF with AL was demonstrated by anti-CyclinB2 immunofluorescence in prophase oocytes, in which AL are positioned close to the vegetal surface. Green fluorescent protein-CyclinB2 expressed in oocytes also localized at AL. These data suggest that inactive MPF associates with nuclear envelope components just before activation. This association may explain why nuclei and centrosomes stimulate MPF activation and provide a mechanism for targeting of MPF to some of its key substrates.
Beckhelling
Clare
C
Unité Mixte Recherche 7009, Centre National de la Recherche Scientifique/Université Paris VI, Observatoire Oceanologique de Villefranche sur Mer, 06234, Villefranche sur Mer, France.
Chang
Patrick
P
Chevalier
Sandra
S
Ford
Chris
C
Houliston
Evelyn
E
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D016200:Maturation-Promoting Factor / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D009685:Nuclear Envelope / Q000378:metabolism; D009865:Oocytes / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014020:Tissue Extracts / Q000737:chemistry; D014982:Xenopus laevis / Q000502:physiology*; D020687:cdc25 Phosphatases / Q000378:metabolism
D018797:Cell Cycle Proteins; D019926:Cyclin B; D011993:Recombinant Fusion Proteins; D014020:Tissue Extracts; D016203:CDC2 Protein Kinase; D016200:Maturation-Promoting Factor; D020687:cdc25 Phosphatases