Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and colocalization studies in S. cerevisiae .

Autor:	Oppenheimer KG; Department of Biological Sciences, University of Pittsburgh, PA 15260., Hager NA; Department of Biological Sciences, University of Pittsburgh, PA 15260., McAtee CK; Department of Biological Sciences, University of Pittsburgh, PA 15260., Filiztekin E; Department of Biological Sciences, University of Pittsburgh, PA 15260., Shang C; Department of Biological Sciences, University of Pittsburgh, PA 15260., Warnick JA; Department of Biological Sciences, University of Pittsburgh, PA 15260., Bruchez MP; Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA 15213., Brodsky JL; Department of Biological Sciences, University of Pittsburgh, PA 15260., Prosser DC; Department of Biology, Virginia Commonwealth University, Richmond, VA 23284., Kwiatkowski AV; Department of Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261., O'Donnell AF; Department of Biological Sciences, University of Pittsburgh, PA 15260.
Jazyk:	angličtina
Zdroj:	Molecular biology of the cell [Mol Biol Cell] 2024 Jul 01; Vol. 35 (7), pp. mr5. Date of Electronic Publication: 2024 May 29.
DOI:	10.1091/mbc.E24-04-0174
Abstrakt:	Spatial and temporal tracking of fluorescent proteins (FPs) in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active FPs fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis - and trans -acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae . Competing Interests: Conflict of interest statement: The authors declare no conflicts of interest. The authors declare no competing financial interests.
Databáze:	MEDLINE
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