Separate SCF(CDC4) recognition elements target Cdc6 for proteolysis in S phase and mitosis

Abstract

The Cdc6 DNA replication initiation factor is targeted for ubiquitin-mediated proteolysis by the E3 ubiquitin ligase SCF(CDC4) from the end of G1phase until mitosis in the budding yeast Saccharomyces cerevisiae. Here we describe a dominant-negative CDC6 mutant that, when overexpressed, arrests the cell cycle by inhibiting cyclin-dependent kinases (CDKs) and, thus, prevents passage through mitosis. This mutant protein inhibits CDKs more efficiently than wild-type Cdc6, in part because it is completely refractory to SCF(CDC4)-mediated proteolysis late in the cell cycle and consequently accumulates to high levels. The mutation responsible for this phenotype destroys a putative CDK phosphorylation site near the middle of the Cdc6 primary amino acid sequence. We show that this site lies within a novel Cdc4-interacting domain distinct from a Cdc4-interacting site identified previously near the N-terminus of the protein. We show that both sites can target Cdc6 for proteolysis in late G1/early S phase whilst only the newly identified site can target Cdc6 for proteolysis during mitosis.

Fig. 1. (A) W303-1a or W303-1aΔsic1 were transformed with pRS426 (Christianson et al., 1992), pRS426 GAL1,10–CDC6 or pRS426 GAL1,10–CDC6-d2 and grown on glucose (no expression from the GAL1,10 promoter) or galactose to induce expression of the CDC6 gene. Expression of CDC6-d2 but not CDC6 resulted in an inhibition to growth. As can be seen on the right-hand half of each plate this effect was not dependent on SIC1. (B) W303-1a with an integrated copy of either (i) GAL1,10–CDC6 or (ii) GAL1,10–CDC6-d2 was grown in YPRaf until mid-log phase. At this point the cells were transferred into YPGal and allowed to continue cycling. Samples were taken every hour and processed for flow cytometry.

Fig. 2. (A) YGP28 (vector), YGP24 (GAL1,10–CDC6) or YGP25 (GAL1,10–CDC6-d2) cells were grown in YPRaf to mid-log phase and then transferred into either fresh YPRaf or YPGal and allowed to grow for 6 h. Three microlitres from each culture were examined by phase contrast microscopy. (B) YGP28 (lane 3), YGP24 (lane 4) and YGP25 (lane 5) cells were grown in YPRaf to mid-log phase and then blocked in G₂/M with nocodozole. Once 90% of the cells had a large budded phenotype they were transferred into YPGal also containing nocodazole. After a further 2.5 h, samples were taken and processed for immunoblotting to examine the phosphorylation status of the DNA polymerase α subunit p86/91. Control samples to visualize the usual pattern of p86/91 phosphorylation in G₁ (lane 1) and G₂/M (lane 2) cells are also shown.

Fig. 3. YGP24 (GAL1,10–CDC6) and YGP25 (GAL1,10–CDC6-d2) cells were grown in YPRaf until mid-log phase, the cultures were then split into two and blocked with either nocodazole (A) or α factor (B). After blocking GAL1,10–CDC6 or GAL1,10–CDC6-d2 was induced by the addition of galactose. Thirty minutes later, transcription from the GAL1,10 promoter and translation were repressed (T = 0). Further samples were taken at the times indicated and processed for immuno blotting. LC is the loading control showing a region of Ponceau-S-stained membrane corresponding to 55–66 kDa. (C) YP25 cells were grown in YPRaf and blocked with α factor to synchronize them. Once the cells were blocked galactose was added to induce expression of GAL1,10–CDC6-d2. After 30 min the cells were washed and released from the block into YPRaf/Gal. Samples were taken at the time of release and every subsequent 10 min to be processed for immuno blotting and to assess the budding index.

Fig. 4. (A) A schematic diagram to show the relative positions of the eight ^S/_TP motifs in Cdc6. The amino acid sequence around sites 7 and 8 is shown and the arrow denotes a T to M substitution at amino acid position 368 caused by the mutation in CDC6-d2. Amino acid residue numbers are at the top of the diagram; the region of similarity in various AAA⁺ family members is indicated as a grey box (Perkins and Diffley, 1998; Neuwald et al., 1999). (B) GAL1,10 promoter shut-off experiments with cells blocked in G₂/M by nocodazole and expressing either GAL1,10–CDC6-S354A (phosphorylation site 6) or GAL1,10–CDC6-S372A (phosphorylation site 8). Transcription and translation were repressed and a sample was taken (T = 0) to be processed for immunoblotting. Subsequent samples were taken at the times indicated.

Fig. 5. (A) Similarity of a Cdc4-interaction domain in the N-terminus of Cdc6 with sequence around the mutation in Cdc6-d2. The numbers denote the amino acid in Cdc6 at the start and finish of the two sequences. (B) Two-hybrid analysis with these two regions of Cdc6.

Fig. 6. (A) The requirement for the ^S/_TP motifs in the N-terminal fragment of Cdc6 was tested in a two-hybrid assay with Cdc4p. The top row shows the two-hybrid interaction between the N-terminus of Cdc6 and Cdc4p described previously. In the second row the N-terminus but with the ^S/_TP motifs mutated to A[^S/_TP(1–4)A] does not interact with GAD-Cdc4. (B) Promoter shut-off experiments with Cdc6-^S/_TP(1–4)A in cells blocked in G₁ or G₂/M show that these motifs are not required for instability in G₁ or G₂/M. However, this instability is dependent on CDC4 (C) in G₂/M since Cdc6-^S/_TP(1–4)A is stable in cdc4ts cells blocked in nocodazole. (D) Cdc6ΔNT is more stable than the full-length protein. It is, however, degraded at a slower rate as can be seen above in a promoter shut-off in G₂/M blocked cells. Mutation of the remaining ^S/_TP(5–8)A in this truncated protein stabilizes it.

Fig. 7. (A) YLD66 cells grown in YPRaf were synchronized with α factor. Once the cells were blocked, galactose was added to induce the Cdc6-^S/_TP(1–4)A protein. After a further 30 min the cells were released from the block into YPRaf/Gal. Samples were taken at the time of release (T = 0) and every 10 min after for a duration of 90 min to assess the budding index and for immunoblotting to detect levels of Cdc6-^S/_TP(1–4)A. (B) A similar experiment was performed with strain YLD69 [GAL1,10–CDC6-^S/_TP(1–4)A,d2].