The Neurospora clock protein FREQUENCY (FRQ) inhibits its transcriptional activator WHITE COLLAR COMPLEX (WCC) in a negative feedback loop and supports its accumulation in a positive loop. We show that positive feedback is a delayed effect of negative feedback underlying the same post-translational mechanisms: DNA-binding-competent active WCC commits rapidly to degradation. FRQ-dependent phosphorylation of WCC, which interferes with DNA binding (negative feedback), leads to reduced turnover and slow accumulation of newly expressed WCC (positive feedback). When DNA binding of WCC is compromised by mutation, its accumulation is independent of FRQ. Cycles of FRQ-dependent inactivation and PP2A-dependent reactivation of WCC occur in the minute range and are coupled to obligate rapid cycles of nucleo-cytoplasmic shuttling. WCC shuttling and activity cycles are modulated by FRQ in circadian fashion. We show here that FRQ supports negative and positive limbs of the clock by the same molecular mechanisms. Positive feedback (FRQ-dependent accumulation of WCC) is a delayed consequence of negative feedback (FRQ-dependent inactivation of WCC) rather than a mechanistically distinct feedback loop: WCC is active when FRQ is low or absent. Our data indicate that DNAbinding-competent, active WCC is unstable and rapidly turned over. FRQ-dependent phosphorylation of WCC interferes with DNA binding. This results in reduced turnover and allows accumulation of newly expressed WCC. Inactivation and reactivation of WCC are coupled to cycles of nucleo-cytoplasmic shuttling. We show that PP2A/RGB-1 activity is cytoplasmic, and hence passage of the WCC through the cytosol is obligatory for reactivation. Surprisingly, phosphorylation and shuttling cycles occur in the range of minutes and are modulated by FRQ in circadian fashion. Results and DiscussionWe investigated whether FRQ affects turnover of the WCC. In wild type, WCC is stable in constant darkness (DD) but turned over rapidly in constant light (LL) (Lee et al. 2000). To assess the influence of FRQ on WCC turnover, cultures of wild type and frq 9 , a mutant strain harboring a nonfunctional frq allele, were grown in LL. Turnover kinetics were then measured in the presence of cycloheximide (CHX). Degradation of WCC was substantially faster in frq 9 (t 1/2 ∼ 2.4 h) than in wild type (t 1/2 ∼ 4.2 h), demonstrating that FRQ stabilizes the light-activated WCC (Fig. 1A,F).In the negative feedback loop, FRQ promotes phosphorylation of WCC, which leads to its inactivation (Schafmeier et al. 2005). To investigate whether WCC activity affects its stability, we analyzed turnover of WCC in the wc-2G3 strain (Linden et al. 1997). wc-2G3 encodes a WC-2 version that lacks the C-terminal Zincfinger (Zn-finger) domain (Fig. 1B) and is henceforth referred to as wc2⌬C. WC-1 is unstable and does not accumulate in the absence of its assembly partner WC-2 (Cheng et al. 2002). WC-1 accumulated in high levels in wc-2⌬C (Fig. 1C), demonstrating that it assembled with the truncated WC-2⌬C. However, the mutan...

The Saccharomyces cerevisiae CLASP (CLIP-associated protein) Stu1 is essential for the establishment and maintenance of the mitotic spindle. Furthermore, Stu1 localizes to kinetochores. Here we show that, in prometaphase, Stu1 assembles in an Ndc80-dependent manner exclusively at kinetochores that are not attached to microtubules. Stu1 relocates to microtubules when a captured kinetochore reaches a spindle pole. This relocation does not depend on kinetochore biorientation, but requires a functional DASH complex. Stu1 at detached kinetochores facilitates kinetochore capturing. Furthermore, since most of the nuclear Stu1 is sequestered by one or a few detached kinetochores, the presence of detached kinetochores prevents Stu1 from localizing the spindle, and therefore from stabilizing the spindle. Thus, the sequestering of Stu1 by detached kinetochores serves as a checkpoint that keeps spindle poles in close proximity until all kinetochores are captured. This is likely to facilitate kinetochore biorientation. In agreement with the findings described above, a kinetochore mutant (okp1-52) that fails to release Stu1 from the kinetochore displays a severe spindle defect upon spindle pole body separation, and this defect can be rescued by destroying the okp1-52 kinetochore.[Keywords: Kinetochore; spindle; Stu1; CLASP; Okp1] Supplemental material is available at http://www.genesdev.org.

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