In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

The separation of sister chromatids in anaphase depends on the dissociation of cohesin from chromosomes. In vertebrates, some cohesin is removed from chromosomes at the onset of anaphase by proteolytic cleavage. In contrast, the bulk of cohesin is removed from chromosomes already in prophase and prometaphase by an unknown mechanism that does not involve cohesin cleavage. We show that Polo-like kinase is required for the cleavage-independent dissociation of cohesin from chromosomes in Xenopus. Cohesin phosphorylation depends on Polo-like kinase and reduces the ability of cohesin to bind to chromatin. These results suggest that Polo-like kinase regulates the dissociation of cohesin from chromosomes early in mitosis.

The initiation of anaphase and exit from mitosis depend on the anaphase-promoting complex (APC), which mediates the ubiquitin-dependent proteolysis of anaphase-inhibiting proteins and mitotic cyclins. We have analyzed whether protein phosphatases are required for mitotic APC activation. In Xenopus egg extracts APC activation occurs normally in the presence of protein phosphatase 1 inhibitors, suggesting that the anaphase defects caused by protein phosphatase 1 mutation in several organisms are not due to a failure to activate the APC. Contrary to this, the initiation of mitotic cyclin B proteolysis is prevented by inhibitors of protein phosphatase 2A such as okadaic acid. Okadaic acid induces an activity that inhibits cyclin B ubiquitination. We refer to this activity as inhibitor of mitotic proteolysis because it also prevents the degradation of other APC substrates. A similar activity exists in extracts of Xenopus eggs that are arrested at the second meiotic metaphase by the cytostatic factor activity of the protein kinase mos. In Xenopus eggs, the initiation of anaphase II may therefore be prevented by an inhibitor of APC-dependent ubiquitination. INTRODUCTIONThe activation of Cdc2 and possibly of other mitotic protein kinases is thought to be largely responsible for the structural reorganization of the cell during prophase and metaphase (reviewed by King et al., 1994;Nigg et al., 1996). These events, in particular chromosome condensation and spindle assembly, are essential prerequisites for the following separation of sister chromatids in anaphase. However, the initiation of anaphase itself and the subsequent exit from mitosis appear to depend on the activation of a multisubunit ubiquitination complex, called the anaphase-promoting complex (APC) 1 in vertebrates and yeast or the cyclosome in clams (Irniger et al., 1995;King et al., 1995;Sudakin et al., 1995;Peters et al., 1996;Zachariae et al., 1996). This complex catalyzes the assembly of polyubiquitin chains on several specific substrate proteins, which targets them for destruction by the 26S proteasome complex (reviewed by Peters, 1994;Coux et al., 1996).The first event in mitosis that is known to depend on the APC is the initiation of sister chromatid separation at the metaphase-to-anaphase transition (Holloway et al., 1993;Irniger et al., 1995). Proteins that have to be destroyed to allow anaphase have recently been identified in budding yeast as the Pds1 protein and in fission yeast as Cut2 but are still unknown in other organisms (Cohen-Fix et al., 1996;Funabiki et al., 1996). The best studied substrates of the APC are the mitotic cyclins, the positive regulatory subunits of Cdc2. Synthesis of cyclins during interphase is necessary to allow activation of Cdc2 and entry into mitosis, whereas APC-mediated degradation of cyclins in anaphase results in inactivation of the kinase (reviewed by King et al., 1994). The degradation of cyclins requires a short sequence element found in the N-terminal portion of mitotic cyclins, called the destruction box (Glotz...

The aim of the present review is to summarize recent progress in identifying substrate binding domains of P-glycoprotein by photoaffinity labeling. Preferred substrate binding regions have been identified using a number of photoaffinity ligands, including anthracyclines, the quinazoline iodoarylazidoprazosine (IAAP), dihydropyridines, taxanes and propafenones. These studies allowed identification of protein regions, which are involved in ligand interaction.