ht bands (resembling a ladder) have been observed only in cdc20- and cdc22-arrested extracts (Fig 1E), suggesting that Rev1 underwent ubiquitindependent proteolysis. Ubiquitination targets the lysine residues of your protein; therefore, we first constructed Rev1dKK, an 110044-82-1 internal deletion mutant (76118) that lacked the lysine-rich region close to the C terminus (Fig 2A). As anticipated, Rev1dKK protein levels were a lot larger than those of Rev1wt, even in unsynchronized circumstances (Fig 2B). Subsequent, we examined the protein degree of Rev1 inside the Rev1dKK mutant in cdc25-synchronized cultures. As shown in Fig 2C, Rev1dKK protein levels peaked at S phase instead of G1 phase. Because cell cycle-dependent oscillation of rev1 mRNA was previously reported [43], the observed protein level oscillation of Rev1dKK probably resulted from regulation from the mRNA level. We also designed a Rev1dK internal deletion mutant (76197) since the Rev1dKK mutant lacked a ubiquitin-binding motif, which can be critical for the function of Rev1. Rev1 protein was 
also elevated inside the Rev1dK mutant when compared with the Rev1wt strain (S2 Fig). These data suggested that Rev1 underwent ubiquitin-dependent proteolysis at G1/S phase.
An internal deletion mutant stabilized Rev1 protein in the S phase. A, Schematic diagram of the fission yeast Rev1 domain structure. Parallelogram, BRCT; rectangle, Y family-conserved area; ellipse, ubiquitin binding motif; hexagon, Lys-rich area. Rev1dK and Rev1dKK are internal deletion mutants lacking the amino acids from 761 to 818 and 761 to 797, respectively. B, The Rev1dKK mutant stabilized Rev1 protein. Flag-tagged rev1wt and rev1dKK cells had been grown, and entire cell extracts were prepared by the boiling approach. The upper panel shows a western blot of Rev1 protein, plus the lower panel shows CBB staining in the membrane as a loading control. The left lane represents Rev1wt, plus the correct lane represents Rev1dKK. C, Rev1dKK protein was steady in S phase. Time course samples with the rev1dKKflag cdc25 strain were prepared. The samples had been taken every 30 min soon after the release. The lower panels show the expression patterns of Rev1dKK, Cdc13, and Cdc2.
Ubiquitin-dependent proteolysis happens by way of the activity of proteasome complexes [44, 45]. Therefore, we subsequent analyzed Rev1 protein levels in proteasome-deficient conditions. First, we employed random mutagenesis to construct the mts2/rpt2 temperature-sensitive mutant, which disrupted the function of a proteasomal subunit [46] following appropriate adjustments in temperature. The protein level of Rev1 was then examined at the restrictive situation in the temperature-sensitive mutant. The protein levels of Cdc13, which can be controlled by proteasome-dependent proteolysis [47], and Rev1 were elevated right after mts2-U31 mutant cells had been arrested (Fig 3A). In contrast, the protein levels on the Rev1dK mutant 21593435 did not increase soon after mts2-U31-dependent cell cycle arrest (Fig 3B). The apparent reduce in the protein degree of Rev1dK resulted in the rising temperature and cell cycle arrest in M phase attributable to mts2-U31. To further confirm the contribution from the proteasome to Rev1 protein levels, we also constructed mts3/ rpn12 [48] temperature-sensitive mutants. Similarly, Rev1 protein levels increased in mts3-U32 cells at the restrictive temperature (Fig 3C), and also the volume of Rev1dK did not improve below the same conditions (Fig 3D). These data clearly indicated that Rev1 protein levels were controlled by proteasomal degradation.
Rev1 pr
