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

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 GBPmCherry-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.

F-box proteins Pof3 and Pof1 regulate Wee1 degradation and mitotic entry in fission yeast

“…Therefore, we expected that further elevated Wee1 in pof3Δ and pof1-6 mutants would lead to lethality as a result of strong G 2 arrest. To test this possibility, we first constructed a series of yeast strains expressing different levels of Wee1 under promoters of various strengths, including nmt promoter variants (Basi et al, 1993;Maundrell, 1990) and constitutively expressing adh promoters, in which P adh11 , P adh21 and P adh81 are weaker versions of P adh1 in the order P adh1 >P adh11 >P adh21 >P adh81 (Chen et al, 2017;Kawashima et al, 2007;Tada et al, 2011;Yokobayashi and Watanabe, 2005). We obtained strains carrying genomically integrated wee1 + driven under different promoters, except with adh11 and adh1 promoters (Fig.…”

“…To generate strains expressing wee1 + under nmt promoters at the leu1 + locus, the coding sequence of wee1 + was amplified from yeast genomic DNA using the polymerase chain reaction (PCR) and subcloned into the vector pJK148-P nmt1 ::leu1 + (Chen et al, 2017) using SalI/BamHI sites, which resulted in the construction of the final vector pJK148-P nmt1 -wee1 + :: leu1 + . The plasmids of pJK148-P nmt41 -wee1 + ::leu1 + and pJK148-P nmt41 -wee1 + ::leu1 + with weaker nmt promoters were constructed by Quikgene site-directed mutagenesis as previously described (Chen et al, 2017).…”

“…To construct strains expressing wee1 + under different strength adh promoters at the lys1 + locus, the PCR-amplified coding sequence of wee1 + was subcloned into a series of pUC119-based vectors carrying sequences corresponding to different strength adh promoters (Chen et al, 2017) using SalI/BamHI sites. This procedure generated four vectors: pUC119-P adh81 -wee1 + ::hphMX6::lys1*, pUC119-P adh21 -wee1 + ::hphMX6::lys1*, pUC119-P adh11 -wee1 + ::hphMX6::lys1* and pUC119-P adh1 -wee1 + ::hphMX6::lys1*.…”

Protein binders and their applications in developmental biology

“…diffusible growth factors: Matsuo and Kimura-Yoshida, 2014), the functional implications or importance of protein localization are more difficult to assess. Recently, however, protein binders fused to localization domains have been successfully used to relocalize POIs in a controlled manner, and have thus allowed the systematic study of protein localization/mislocalization during animal development (Berry et al, 2016;Chen et al, 2017;Harmansa et al, 2017;Rothbauer et al, 2008). In these studies, protein relocalization is achieved by fusing a GFP-binding nanobody to a protein or a protein domain that localizes to a defined position within the cell or the tissue.…”

Tools for retargeting proteins within Aspergillus nidulans

“…To generate the chromatin-NPC bridge we used the GFP Binding Protein (GBP) system [ 2 ]. The GFP-GBP system has been developed for use in human cells and has also been successfully employed in plants, fission yeast and prokaryotes but has had limited application in filamentous fungi [ 3 – 6 ]. This system is based on a genetically encoded Llama single chain antibody against GFP [ 2 , 3 ].…”