Bioinformatic analysis of the protein sequence of Tpz1 using SUMOplotTM Analysis
Program identified a putative SUMOylation site at lysine 242 with the sequence VKQE conforming to the general ΨKX(D/E) SUMO consensus. To determine whether the predicted SUMOylation site in Tpz1 is conserved, Tpz1 protein sequences from all four Schizosaccharomyces species were aligned using Clustal Omega and the output was generated using Jalview. The alignment revealed that the other three fission yeast strains (S. cryophilus, S. octosporus and S. japonicas) have a potential SUMOylation site at a similar location (Figure 2.1A). This suggested that SUMOylation of Tpz1 might be a highly conserved post- translational modification in fission yeast. To further investigate the role of Tpz1 SUMOylation, a mutation at the putative Lys242 was introduced in the chromosomal copy of the tpz1+ gene. Firstly, a yeast integrative vector pSpTpz1- Ui, containing the wild-type tpz1 ORF, ura4+ marker (for selection in yeast), AmpR ORF (for plasmid selection in bacteria) and pUC origin of replication (for plasmid propagation in bacteria), was constructed. Plasmid pEXA-Tpz1KR containing a fragment of the tpz1 ORF with lysine 242 mutated to arginine and a unique HaeIII restriction site in the fragment was synthesised by Eurofins MWG Operon. The HaeIII restriction site was specifically introduced to enable screening for the tpz1 allele with the K242R mutation as compared to the wild-type that lacks this site at that location. A 210bp AflII-MluI fragment from pEXA-Tpz1KR was sub-cloned into the same sites in the linearized pSpTpz1-Ui vector to generate pSpTpz1-K242R-Ui, an integrative vector containing the mutant tpz1 gene.
Plasmid pSpTpz1-K242R-Ui was linearized with NdeI or XbaI restriction enzyme and transformed into a ura4 mutant strain for integration at the tpz1 locus. The integration process results in a partial duplication of the tpz1 ORF in the genome, though only one copy will be full-length and under tpz1 promoter control. Following plasmid integration, selection for rare recombination events between the two copies of the tpz1 ORF using 5-FOA media resulted in recovery of strains bearing a single complete tpz1 ORF and devoid of any plasmid sequences (Figure 2.1B). In order to identify clones that retained the tpz1- K242R allele as opposed to the wild-type version, the tpz1 locus of the recombined strains was amplified by PCR using the primers tpz1-f1-Sp and tpz1-b4-Sp and the resultant 819 bp PCR product was digested with HaeIII restriction enzyme. Whereas the PCR product from the strains containing the wild-type tpz1 locus would remain undigested, the one with K242R mutation would be digested to give 400 and 419 bp fragments, identified by agarose gel electrophoresis (Figure 2.1C).
The strains containing the tpz1-K242R allele were Flag-tagged at the C-terminus, similar to the wild-type strain, using pSpTpz1-G10F106 vector. The pSpTpz1- G10F106 vector contained a C-terminal fragment of the tpz1 ORF (567 bp) fused to an array of 10 flag epitope sequences through a linker of 8 glycine residues. When linearised, this allows integration at the C-terminal end of Tpz1, in frame with the ORF, facilitating antibody recognition of the expressed protein. Protein extracts were prepared using equal number of log phase cells in order to assess relative expression of the Flag-tagged Tpz1 in wild-type and mutant strains by anti-flag western blotting. The expression of Tpz1 in the mutant strain was observed to be similar to wild-type (data not shown).
Histidine tagged SUMO variant was introduced in the strains containing the wild- type tpz1 and the tpz1-K242R allele, both with a C-terminal flag tag on Tpz1, by transforming the plasmid pREP41-hisSUMO plasmid. Plasmid pREP41-hisSUMO was kindly provided by Felicity Watts and contains a 6xHis tag fused to the N- terminal region of pmt3 ORF with the expression of the protein under the control of a thiamine repressible nmt41 promoter. To repress the expression of the episomal His-tagged SUMO gene, the transformation mixes were plated on the appropriate selective media containing 15mM thiamine. Transformants were initially grown in an appropriate liquid media containing 15mM thiamine and then washed and transferred to the media lacking thiamine to induce the expression of 6xHis-SUMO. The cells were collected immediately after changing the media and then after 22hours induction of histidine-tagged SUMO gene. Ni-NTA affinity chromatography under denaturing conditions was used for bulk purification of the His-tagged SUMO and hence SUMO conjugated Tpz1 from the whole cell extracts. This method minimizes deconjugation and excludes noncovalent interactions (Ulrich, & Davies 2009). Protein extracts were analysed by SDS-PAGE and western blotting using anti-flag primary antibody. As expected, the western blot analysis revealed the presence of a lower mobility band in the affinity-purified fraction, following 22hour induction of the histidine-tagged SUMO gene, in the strain with a flag-tagged copy of wild type tpz1 (Figure 2.1D, lanes 1-4). This further confirmed the SUMOylation of Tpz1, as observed previously. The lower mobility band was not recovered in the affinity-purified fraction in the strain with a flag-tagged copy of mutant tpz1-K242R allele (Figure 2.1D, lanes 7-10). This data indicated that SUMO covalently links to Tpz1 at Lysine 242.
Figure 2.1: SUMOylation of Tpz1 at Lys242.
(A) Schizosaccharomyces species S. pombe, S. cryophilus, S. octosporus and S. japonicas was generated using alignment tool in Jalview. The SUMOylation consensus (ΨKX(D/E)) is highlighted in pink with the target lysine in red. (B) The integrative plasmid pSpTpz1-K242R-Ui was linearized with NdeI present within the Tpz1 orf and transformed into the S. pombe genome. Integration of the plasmid through recombination with endogenous tpz1 resulted in two copies of tpz1 and introduced the ura4+ marker into the yeast genome. The ura4+ marker was then counter-selected for using FOA containing growth media resulting in a second recombination event. The ura4 marker was ejected along with one copy of the tpz1 gene. Depending on the site of crossover between the two copies of the tpz1 genes either tpz1 gene with mutation or the wild-copy of the gene was retained. (C) PCR analysis of two putative mutant colonies alongside positive and negative controls (pSpTpz1-K242R-Ui uncut and cut using HaeIII). (D) Fission yeast cells contained a 10x-Flag epitope tag at the Tpz1 C-terminus and a plasmid expressing a 6x-histidine tagged version of SUMO (Pmt3) under control of the nmt41 promoter, which is repressed by thiamine and slowly induced upon thiamine withdrawal. At 22 h after thiamine removal, whole-cell protein extracts (WCE) were prepared. SUMO-modified proteins were then affinity-purified (A-P) on Ni- agarose beads under denaturing conditions. Both whole-cell extract and affinity-purified material
S. cryophilus S. octosporus S. japonicus S. pombe NSPDNTQIKEEEGVEF---YSWSSSPNL-
SVSETPEVKQEDNDEDLDAYSWSSSTDS-261
NLPDDIHVKEEEQMEI---YSWSSSPEP-258
73
EDEDVPVIKHEPDVSAAD-FSWSSSPDT-271
242 A B Tpz1-10xFlag tpz1+ tpz1+ tpz1-K242R tpz1-K242R tpz1+ tpz1-K242R 1 1 2 3 4 5 6 7 8 2 3 4 5 6 7 8 9 10 SUMO-6xHis induction - - + + + + - - + +
WCE A-P WCE A-P WCE A-P WCE A-P WCE A-P
+ + + + - - + + + + Flag epitopes 75 kD 100 kD C D Tpz1-K242R ORF
Tpz1 ORF genomic locus
genomic locus AmpR f1(+) pUC Ori ura4+ Plasmid controls PCR products Tpz1-K242R ORF - + - + - + - + 500 400 1000 HaeIII bp HaeIII NdeI XbaI pSpTpz1-K242R-Ui
integration at the wild-type locus
Desired product of recombination
transformation of linearised plasmid in yeast
Recombination * * * * // // // // // // // = K242R mutation