Fig. 2. Identification of a lysine residue modified by NEDD8 in Pcu1 and Pcu4 in S.pombe. (A) Amino acid sequences of the C-terminal region of six members of the human cullin family, Hs-Cul-1, -2, -3, -4A, -4B and -5, three members of the fission yeast cullin family, Pcu1, Pcu3 and Pcu4, and the budding yeast Cdc53 are shown. The numbers shown are the last residue numbers of the peptides in the respective proteins. Gaps (shown by dots) are inserted to maximize sequence homologies. Amino acids conserved in half or more of these cullins are boxed in black. The arrowheads indicate two lysine residues conserved in all members of the cullin family proteins. Pcu1^K713R, Pcu4^K680R and Pcu4^K710R indicate that lysine at positions 713, 680 and 710 in Pcu1 and Pcu4, respectively, was replaced by arginine. (B) Wild-type cells containing pREP41-HA-pcu1⁺ and pREP81-Myc-ned8⁺ were grown in minimal medium without (de-repressed, lanes 1 and 3–6) or with (repressed, lane 2) thiamine. Cell extracts were immunoprecipitated with anti-HA antibody or non-immune mouse IgG. Cell extracts (lanes 1 and 2) and immunoprecipitates (lanes 3–6) were separated by SDS–PAGE, followed by immunoblotting using anti-HA or anti-Myc antibody. An asterisk indicates HA-Pcu1 ligated by Myc-NEDD8. (C) After [³⁵S]FLAG-Pcu4 was synthesized in a reticulocyte lysate transcription/translation system in the presence or absence of unlabeled GST–Ub, GST–NEDD8 or GST–NEDD8^ΔG76 (ΔG76) lacking the C-terminal glycine residue, samples of the resultant translational products were subjected directly to SDS–PAGE in the presence of DTT and autoradiographed (lanes 1–4). Two Pcu4 variants, FLAG-Pcu4^K680R and FLAG-Pcu4^K710R, were synthesized in vitro in the presence of GST–NEDD8 and analysed by the same method (lanes 5 and 6). An asterisk indicates [³⁵S]FLAG-Pcu4 ligated by GST–NEDD8. (D) Wild-type cells containing pREP41-HA-pcu1⁺ (Wt) or pREP41-HA-pcu1^K713R (K713R) (upper panel), or pREP81-FLAG-pcu4⁺ (Wt) or pREP81-FLAG-pcu4^K680R (K680R) (lower panel) were grown in minimal medium with or without thiamine. Proteins were detected by immunoblotting using anti-HA or anti-FLAG antibody. Asterisks indicate FLAG-Pcu4 or HA-Pcu1 ligated by NEDD8. Note that the nmt1 promoter on pREP41 is ∼12-fold more active than that on pREP81 and that the expressed levels of the mutants FLAG-Pcu4^K680R and Myc-Pcu1^K713R, which did not form a linkage to NEDD8, were roughly similar to those of their wild types.

Fig. 4. pcu1^K713R loses the ability to complement the lethality of Δpcu1 cells. (A) Spores (5 × 10³) derived from the IO100 (pcu1::ura4⁺/pcu1⁺) diploid strain containing [pREP41-Myc-pcu1⁺ LEU2] or [pREP41-Myc-pcu1^K713R LEU2] were germinated and grown on minimal medium plates lacking uracil and leucine in the presence (upper panel, repressed) or absence (lower panel, de-repressed) of thiamine (B1). (B) Δpcu1 cells and Δpcu1 cells expressing Myc-Pcu1^K713R, germinated from spores, are shown. Bar, 10 µm.

Fig. 6. Accumulation of Rum1 in NEDD8-depleted cells. Δned8⁺ cells containing pREP81- ned8⁺ were grown in minimal medium with thiamine for the indicated time at 32°C. (A) DNA content and cell size were measured by a flow cytometer. (B) Cell extracts were separated by SDS–PAGE, followed by immunoblotting using anti-Rum1 or anti-tubulin antibody.

Fig. 1. Functional analysis of the NEDD8-ligating system in fission yeast. (A) Amino acid comparison of S.pombe (Sp) NEDD8 (DDBJ/EMBL/GenBank accession No. AJ003818), Uba3 (SWISS-PROT accession No. Q09765) and Ubc12 (DDBJ/EMBL/GenBank accession No. AL031532, SPC777.10C) with their human homologues (Hs). The identical amino acids are boxed in black. The asterisks in Uba3 and Ubc12 indicate the putative active cysteine residues. The arrow in NEDD8 indicates the processing site. (B) Disruption of ned8⁺, uba3⁺ and ubc12⁺ genes. The coding sequences in exons of the ned8⁺, uba3⁺ and ubc12⁺ genes are shown by solid boxes. For gene disruption, the indicated DNA fragments were replaced by a PCR-generated fragment containing the ura4⁺ marker gene. (C) After tetrad analysis of diploids heterozygous for ned8, uba3 and ubc12, inviable Ura⁺ cells that had germinated from each of the spores were observed under a phase-contrast microscope (upper panel). Note that in SpUba3 and SpUbc12, a part of the cells derived from a microcolony is shown. To observe the defective phenotype by DAPI staining, heterozygous diploids were sporulated and treated with glusulase to kill non-sporulating cells. Spores were grown in minimal medium lacking uracil at 30°C for 24 h then stained with DAPI (lower panel). Bar, 35 µm (upper panels) and 10 µm (lower panels).

Fig. 3. Preferential modification of Pcu1 by NEDD8 in the SCF^Pop1 complex. (A) Wild-type cells (lanes 1 and 4) or cells in which chromosomal pcu1⁺ and pop1⁺ had been replaced by the epitope-tagged genes pop1⁺-HA₃ (lanes 2 and 5), pcu1⁺-Myc₁₃ (lanes 3 and 6) or pop1⁺-HA₃ and pcu1⁺-Myc₁₃ (lanes 7 and 8) were grown in YE. Cell extracts were immunoprecipitated (IP) with anti-HA antibody. The cell extracts (lanes 1–3 and 7) and IP-proteins (lanes 4–6 and 8) were separated by SDS–PAGE followed by immunoblotting with anti-HA, anti-Myc antibody. (B) Fission yeast cells containing the integrated pop1⁺-HA₃ and pREP41-Myc-Pcu1^K713R were grown in minimal medium without thiamine for 20 h at 32°C. Cell extracts were immunoprecipitated (IP) with anti-HA antibody. The cell extracts and immunoprecipitated proteins were separated by SDS–PAGE and followed by immunoblotting with anti-HA, anti-Myc antibody. Asterisks indicate truncated Pop1, which may be produced by proteolytic digestion or synthesized from a different translation initiation site.

Fig. 5. Stabilization of Rum1 in the Pcu1^K713R-overexpressed cells. Fission yeast cells (SKP401) containing pREP41-Myc-pcu1^K713R were grown in minimal medium without thiamine for the indicated time at 32°C. (A) DNA content and cell size were measured by a flow cytometer. (B) Cells were stained with DAPI. Bar, 10 µm. (C) Cell extracts were separated by SDS–PAGE, followed by immunoblotting using anti-Myc, anti-Rum1 or anti-tubulin antibody. (D) Northern blot analysis was carried out using rum1 or his3 cDNA as a probe. (E) Wild-type cells containing pREP42-rum1⁺-HA/His6 and pREP41-Myc-pcu1⁺ (lanes 1–4), or pREP42-rum1⁺-HA/His6 and pREP41-Myc-pcu1^K713R (lanes 5–8) were grown in minimal medium without thiamine for 22 h. At time 0, thiamine was added to repress the nmt1 promoter. After the indicated times, cell extracts were prepared, separated by SDS–PAGE, then subjected to immunoblotting using anti-HA or anti-tubulin antibody (upper panels). The levels of Rum1 were quantified densitometrically, and the relative amounts of remaining Rum1 are shown (lower panel).

Fig. 7. pcu4^K680R loses the ability to complement the defective phenotypes of Δpcu4 cells. (A) Disruption of the pcu4⁺ gene. Almost the entire ORF of the pcu4⁺ gene (DDBJ/EMBL/GenBank accession No. Z99260, SPC3A11.08) was deleted by use of a PCR-generated fragment containing the ura4⁺ marker. (B) Tetrad analysis of diploids heterozygous for pcu4. (C) Δpcu4 cells were stained by DAPI as described in Figure 1C. Ungerminated spores are marked with an arrow. Bar, 10 µm. (D) Δpcu4 cells containing pREP81-FLAG-pcu4⁺ or pREP81-FLAG-pcu4^K680R were grown in minimal medium without thiamine, then observed under a phase contrast microscope. Bar, 10 µm. (E) Δpcu4 cells containing pREP81-FLAG-pcu4⁺ or pREP81-FLAG-pcu4^K680R were grown on a minimal plate without thiamine for 4 days at 30°C.