Many of the proteins that regulate transit through the cell cycle have been characterized. Xnf7, identified in extracts of Xenopus eggs, binds to the anaphase-promoting complex/ cyclosome (A PC/C). To elucidate the function of this protein, studies have been undertaken in which Xnf7 either has been depleted from extracts using an antibody raised against it or has been augmented in the extracts through addition of extra Xnf7. The consequences on transit through mitosis were then assessed.

a. Xenopus egg extracts, arrested in metaphase, were either depleted of Xnf7 or were mock depleted (subjected to the same treatment as the first sample but without Xnf7 antibody), then released from metaphase arrest by addition of Ca²⁺. Aliquots of the extract were then sampled at various times after Ca²⁺ addition and the amounts of mitotic cyclin determined, as shown on the Western blot below. What information do these data provide about a possible function for Xnf7?

b. In additional studies, exogenous Xnf7 was added to Xenopus egg extracts, arrested at metaphase, so that the total amount of this protein in the extracts was higher than normal. The extracts, released from arrest by Ca²⁺ addition, were then assessed at various times after release for mitotic cyclin ubiquitinylation (cyclin-Ub conjugates). What is the rationale for examining ubiquitinylation? Using the following figure, determine what information these studies add beyond that obtained from part (a).

c. The spindle checkpoint pathway prevents cells with unattached kinetochores from proceeding into anaphase. Thus cells in which this checkpoint has been activated do not enter anaphase and do not degrade mitotic cyclin. Nocodazole, a drug that prevents microtubule assembly, can be used to activate the spindle checkpoint. Cells in nocodazole become arrested in early mitosis because they cannot form a spindle, and so all kinetochores remain unattached. To determine if Xnf7 is required for a functional spindle checkpoint, Xenopus egg extracts, arrested in metaphase, were subjected to various protocols (see the following figure): untreated (no nocodazole) or treated with nocodazole and either mock depleted (preimmune) or immunodepleted of Xnf7 (a-Xnf7). The extracts were then treated with Ca²⁺ to overcome arrest, and aliquots of the extracts were assessed at various times for mitotic cyclin, as shown on the Western blot below. What can you conclude about Xnf7 from these data?

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Time (min) o 15 30 40 10 20 60 Mock depleted Xnf7 depleted