McAtee, C. K. | Search bioRxiv

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↗

bioRxiv2021

Alpha-arrestins Aly1 and Aly2 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis

Phosphatidylinositol (PI) is an essential phospholipid and critical component of membrane bilayers. The complete deacylation of PI by phospholipases of the B-type leads to the production of intracellular and extracellular glycerophosphoinositol (GPI), a water-soluble glycerophosphodiester. Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters that resides at the plasma membrane in yeast. Once internalized, GPI can be degraded to produce inositol, phosphate and glycerol, thereby contributing to reserves of these building blocks. Not surprisingly, GIT1 gene expression is controlled by nutrient balance, with limitation for phosphate or inositol each increasing GIT1 expression to facilitate GPI uptake. Less is known about how Git1 protein levels or localization are controlled. Here we show that the -arrestins, an important class of protein trafficking adaptor, regulate the localization of Git1 in a manner dependent upon their association with the ubiquitin ligase Rsp5. Specifically, -arrestin Aly2 is needed for effective Git1 internalization from the plasma membrane under basal conditions. However, in response to GPI-treatment of cells, either Aly1 or Aly2 can promote Git1 trafficking to the vacuole. Retention of Git1 at the cell surface, as occurs in aly1{Delta} aly2{Delta} cells, results in impaired growth in the presences of excess exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of this metabolite or its downstream products leads to cellular toxicity. We further show that regulation of -arrestin Aly1 by the protein phosphatase calcineurin improves both steady-state and ligand-induced trafficking of Git1 when a mutant allele of Aly1 that mimics the dephosphorylated state at calcineurin-regulated residues is employed. Thus, calcineurin regulation of Aly1 is important for the GPI-ligand induced trafficking of Git1 by this -arrestin, however, the role of calcineurin in regulating Git1 trafficking is much broader than can simply be explained by regulation of the -arrestins. Finally, we find that loss of Aly1 and Aly2 leads to an increase in phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole and this alteration is further exacerbated by addition of GPI, suggesting that the effect is at least partially linked to Git1 function. Indeed, loss of Aly1 and Aly2 leads to increased incorporation of inositol label from 3H-inositol-labelled GPI into PI, confirming that internalized GPI influences PI synthesis and indicating a role for the -arrestins in regulating the process.

cell biology↗