Identification of Novel Vesicles in the Cytosol to Vacuole Protein Degradation Pathway

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Abstract
In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor. To examine the possible role of MAP kinase in the in vitro cell cycle, we depleted the extracts of MAP kinase by using anti–Xenopus MAP kinase antibody. Like in the mock-treated extracts, the periodic activation and deactivation of MPF occurred normally in the MAP kinase–depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro. It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase– specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction. We show here that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase–depleted extracts. But, adding recombinant Xenopus MAP kinase to the MAP kinase–depleted extracts restored the spindle assembly checkpoint. These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts. In addition, we show that strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induced mitotic arrest in the extracts. Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro.