Oppenheimer, K. G. | bioRxiv Science

bioRxiv2024

A new series of fluorogen-activating proteins for quantitative protein trafficking and co-localization studies in S. cerevisiae

Spatial and temporal tracking of fluorescent proteins in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (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. SIGNIFICANCE STATEMENT- Quantitative endocytic assays are required to characterize factors that regulate both ligand-dependent and constitutive endocytosis. - We optimize fluorogen-activating proteins (FAPs) technology for use as a live cell imaging probe in yeast that fluoresces in the far-red range for quantitative endocytosis assays. - The FAP tools and approaches generated will facilitate quantitative endocytic and protein recycling assays for yeast cell biologists.

cell biology↗

bioRxiv2022

Hexokinase 2 localizes to the nucleus in response to glucose limitation but does not regulate gene expression

Glucose is the preferred carbon source for most eukaryotes, and the first step in its metabolism is phosphorylation to glucose-6-phosphate. This reaction is catalyzed by a family of enzymes called either hexokinases or glucokinases depending on their substrate specificity. The yeast Saccharomyces cerevisiae encodes three such enzymes, Hxk1, Hxk2 and Glk1. In yeast and mammals, some isoforms of this enzyme are found in the nucleus, suggesting a possible moonlighting function beyond glucose phosphorylation. In contrast to mammalian hexokinases, the yeast Hxk2 enzyme has been proposed to shuttle into the nucleus in glucose replete conditions where it reportedly moonlights as part of a glucose-repressive transcriptional complex. To achieve this role in glucose repression, Hxk2 reportedly binds the Mig1 transcriptional repressor, is dephosphorylated at serine 15 in its N-terminus, and requires an N-terminal nuclear localization sequence (NLS). In this study, we use high-resolution, quantitative, fluorescent microscopy of live cells to determine the conditions, residues, and regulatory proteins required for Hxk2 nuclear localization. In direct contradiction to previous yeast studies, our quantitative imaging demonstrates that Hxk2 is largely excluded from the nucleus under glucose replete conditions but is retained in the nucleus under glucose limiting conditions. Our data show that the Hxk2 N-terminus does not contain an NLS but instead comprises sequences necessary for nuclear exclusion and multimerization regulation. Amino acid substitutions of the phosphorylated residue, serine 15, disrupt Hxk2 dimerization but have no effect on its glucose-regulated nuclear localization. Substitution of alanine at the nearby residue, lysine 13, affects both dimerization and maintenance of nuclear exclusion under glucose replete conditions. Modeling and simulation provide insight into the molecular mechanisms of this regulation. In marked contrast to earlier studies, we find that the transcriptional repressor Mig1 and the protein kinase Snf1 have little effect on Hxk2 localization. Instead, the protein kinase Tda1 is a key regulator of Hxk2 localization. Finally, RNAseq analyses of the yeast transcriptome further dispel the idea that Hxk2 moonlights as a transcriptional repressor, demonstrating that Hxk2 has a negligible role in transcriptional regulation in both glucose replete and limiting conditions. Taken together, our studies provide a paradigm shift for the conditions, residues, and regulators controlling Hxk2 dimerization and nuclear localization. Based on our data, the nuclear translocation of Hxk2 in yeast occurs in glucose starvation conditions, a finding that aligns well with the nuclear regulation of mammalian orthologs of this enzyme. Our findings lay the foundation for future studies of Hxk2 nuclear activity.

cell biology↗