Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP.
J Cell Sci
; 130(5): 1003-1015, 2017 03 01.
Article
in En
| MEDLINE
| ID: mdl-28082423
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Schizosaccharomyces
/
Cytological Techniques
/
Green Fluorescent Proteins
Language:
En
Journal:
J Cell Sci
Year:
2017
Document type:
Article
Affiliation country: