Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP

J Cell Sci. 2017 Mar 1;130(5):1003-1015. doi: 10.1242/jcs.198457. Epub 2017 Jan 12.

Abstract

GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe, we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP-mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP-mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP-mCherry and efficient targeting of Dma1- or Cdc7-GBP-mCherry to the spindle pole body by Sid4-GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale.

Keywords: Adh; Fission yeast; GBP; GBP–GFP high affinity; GBP–mCherry; Nmt; Targeted protein localization.

MeSH terms

  • Base Sequence
  • Cytological Techniques / methods*
  • Genes, Reporter
  • Genetic Vectors / metabolism
  • Green Fluorescent Proteins / metabolism*
  • Luciferases / metabolism
  • Promoter Regions, Genetic / genetics
  • Protein Binding
  • Protein Transport
  • Recombinant Fusion Proteins / metabolism
  • Schizosaccharomyces / metabolism*
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism
  • Spindle Pole Bodies / metabolism

Substances

  • Recombinant Fusion Proteins
  • Schizosaccharomyces pombe Proteins
  • Green Fluorescent Proteins
  • Luciferases