Analysis of a cancer-associated mutation in the budding yeast Nuf2 kinetochore protein

Abstract

The kinetochore is a highly conserved megadalton protein complex that ensures proper chromosome segregation via microtubule attachments. The NDC80 complex is one of the major conserved microtubule binding complexes in the kinetochore. NUF2, a protein within the NDC80 complex, has been identified as a cancer gene candidate because missense mutations, found across different tumor samples, cluster within NUF2's calponin homology domain. In this study, we examined a NUF2 cancer-associated mutation in a simple and well-studied organism, Saccharomyces cerevisiae , to elucidate its effects on cell division. We studied the budding yeast nuf2 ^Q21A mutation with the intention of extrapolating our results to the homologous cancer associated mutation in Homo sapiens NUF2 ^R19H (HsNUF2 ^R19H ). Our studies demonstrate that the nuf2 ^Q21A mutant does not exhibit any growth defects or disrupt kinetochore composition. Additionally, it does not affect the Ndc80 complex's interactions with the Dam1 complex or with the Mps1 kinase. These results indicate that the yeast nuf2 ^Q21A mutant does not cause a significant defect in kinetochore function, and that the role of HsNUF2 ^R19H in cancer will need to be further investigated by directly studying the cancer-associated mutation in human cells.

Figure 1. Characterization of the S. cerevisiae nuf2 ^Q21A mutant

(A) Top: Side-by-side protein structure comparison of the human (in gray; PDB: 2VE7) and S. cerevisiae (in deep teal; PDB: 5TCS) Nuf2 ^CH domains. Structurally conserved residues HsNUF2-R19 and Nuf2-Q21 are highlighted in brown, and both are situated in the same alpha helix of the protein. Inset: Protein structure alignment of amino acids 10-30 of HsNUF2 and amino acids 12-32 of S. cerevisiae Nuf2 . Conserved residues highlighted in brown. Bottom: Jalview Nuf2 protein sequence alignment of the amino acids displayed in the inset. Clustal color scheme denotes colored residues as conserved, and the different colors indicate the biochemical properties of the side chains. (B) Five-fold serial dilution assay of WT (SBY4), NUF2-V5 (SBY22754), and nuf2 ^Q21A -V5 (SBY22924) strains to test temperature and benomyl sensitivity. Strains were grown for 2-3 days on YPD at the indicated temperatures and on different concentrations of benomyl (5 μg/ml and 15 μg/ml). (C) Flag immunoprecipitation of Dsn1-His-Flag from NUF2 - V5 (SBY23066), and nuf2 ^Q21A -V5 (SBY23028) genetic backgrounds analyzed via silver-stained SDS-PAGE. (D) Flag immunoprecipitation of Dad1-Flag from NUF2 - V5 (SBY23067) and nuf2 ^Q21A -V5 (SBY23029) genetic backgrounds were analyzed via silver-stained SDS-PAGE. (E) Five-fold serial dilution assay tested NUF2-V5 (SBY23133), nuf2 ^Q21A -V5 (SBY23073), ask1-2 (SBY1300), NUF2-V5 ask1-2 (SBY23192), and nuf2 ^Q21A -V5 ask1-2 (SBY23190) for temperature and benomyl sensitivity. Strains were grown for 2-3 days on YPD at the indicated temperatures and on different concentrations of benomyl (5 μg/ml and 15 μg/ml).