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Reg1 is a proposed upstream Gpa1 regulator in recycling. (A) Confocal microscopy of wild-type cells co expressing Sec7-GFP and Gpa1-mCherry (top) and Gpa1-GFP and Vps4-mCherry (bottom). (B) Wild-type and rcy1∆ cells expressing Ste3-GFP-DUb with and without Gpa1-mCherry were imaged by confocal microscopy. (C) Ste3-GFP-DUb localization in wild-type cells coexpressing Gpa1 Q323L -mCherry was assessed by fluorescence microscopy. (D) Topscoring mutants from a Gpa1 Q323L mating response screen (Slessareva et al., 2006) are shown, with PI3K mutants (green) and reg1∆ (magenta) highlighted. (E) Confocal microscopy of snf3∆ and reg1∆ cells stably expressing Ste3-GFP-DUb. (F) Quantification of Ste3-GFP-DUb intracellular localizations calculated as an average of population (n = 3) from WT = 68; Gpa1 Q323L = 51; snf3∆ = 83; and reg1∆ = 73 cells. (G) Wild-type (gray) and reg1∆ (red) cells were incubated with rich media containing 40 µM FM4-64 for 8 min before washing and FM4-64 dye efflux measured over time by flow cytometry and plotted by the % of the initial 10 s fluorescence. Scale bar, 5 µm.
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Cell surface protein trafficking is regulated in response to nutrient availability, with multiple pathways directing surface membrane proteins to the lysosome for degradation in response to suboptimal extracellular nutrients. Internalised protein and lipid cargoes recycle back to the surface efficiently in glucose replete conditions, but this traff...
Contexts in source publication
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... localizes to the PM and endomembrane compartments (Sles- sareva et al., 2006;Dixit et al., 2014). Fluorescently tagged Gpa1, which functionally complements gpa1∆ cells (Supplemental Figure S1), colocalizes with the late endosome marker Vps4, and to a lesser degree, the TGN marker Sec7 ( Figure 5A). We found that there was a subtle defect in Ste3-GFP-DUb recycling upon overexpression of Gpa1-mCherry ( Figure 5B), suggesting that both PI3K and Gpa1 levels need to be finely tuned for efficient recycling back to the surface. ...
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... tagged Gpa1, which functionally complements gpa1∆ cells (Supplemental Figure S1), colocalizes with the late endosome marker Vps4, and to a lesser degree, the TGN marker Sec7 ( Figure 5A). We found that there was a subtle defect in Ste3-GFP-DUb recycling upon overexpression of Gpa1-mCherry ( Figure 5B), suggesting that both PI3K and Gpa1 levels need to be finely tuned for efficient recycling back to the surface. Intracellular Ste3-GFP-DUb colocalized with Gpa1-mCherry in both wild type and recycling defective rcy1∆ mutants, suggesting a trafficking block in endosomes from which recycling occurs. ...
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... Ste3-GFP-DUb colocalized with Gpa1-mCherry in both wild type and recycling defective rcy1∆ mutants, suggesting a trafficking block in endosomes from which recycling occurs. Expressing a constitutively active version (Gpa1 Q323L -mCherry) caused more severe defects in Ste3-GFP-DUb recycling ( Figure 5, C and F), again with endosomal reporter colocalizing with Gpa1. Intriguingly, the Gpa1 Q323L screen that revealed Gpa1 couples with PI3K during PtdIns3P production (Slessareva et al., 2006) also demonstrated that the glucose-related phosphatase REG1 is functionally associated Gpa1 ( Figure 5D). ...
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... a constitutively active version (Gpa1 Q323L -mCherry) caused more severe defects in Ste3-GFP-DUb recycling ( Figure 5, C and F), again with endosomal reporter colocalizing with Gpa1. Intriguingly, the Gpa1 Q323L screen that revealed Gpa1 couples with PI3K during PtdIns3P production (Slessareva et al., 2006) also demonstrated that the glucose-related phosphatase REG1 is functionally associated Gpa1 ( Figure 5D). To explore whether the recycling defects associated with Gpa1-PI3K are related to glucose-mediated recycling, we tested recycling in reg1∆ mutants and found that both Ste3-GFPDUb ( Figure 5, E and F) and FM4-64 ( Figure 5G) do not efficiently recycle. ...
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... the Gpa1 Q323L screen that revealed Gpa1 couples with PI3K during PtdIns3P production (Slessareva et al., 2006) also demonstrated that the glucose-related phosphatase REG1 is functionally associated Gpa1 ( Figure 5D). To explore whether the recycling defects associated with Gpa1-PI3K are related to glucose-mediated recycling, we tested recycling in reg1∆ mutants and found that both Ste3-GFPDUb ( Figure 5, E and F) and FM4-64 ( Figure 5G) do not efficiently recycle. No obvious recycling defects were observed in cells lacking the distinct SNF3 glucose-sensing component. ...
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... the Gpa1 Q323L screen that revealed Gpa1 couples with PI3K during PtdIns3P production (Slessareva et al., 2006) also demonstrated that the glucose-related phosphatase REG1 is functionally associated Gpa1 ( Figure 5D). To explore whether the recycling defects associated with Gpa1-PI3K are related to glucose-mediated recycling, we tested recycling in reg1∆ mutants and found that both Ste3-GFPDUb ( Figure 5, E and F) and FM4-64 ( Figure 5G) do not efficiently recycle. No obvious recycling defects were observed in cells lacking the distinct SNF3 glucose-sensing component. ...
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... find that overexpressed Gpa2-mCherry triggered accumulation of Ste3-GFP-DUb in intracellular compartments (Figure 9, A and B). Similarly, cells overexpressing Gpa2-GFP have reduced efflux of FM4-64 from the recycling pathway, when compared with control cells expressing the methionine transporter Mup1-GFP ( Figure 9C and Supplemental Figure S5). In addition to these recycling-specific cargoes, we also used the Gpa2-mCherry overexpression system to reveal prominent defects in recycling of the PM-localized GFP-tagged transporters Mup1 and Can1, which shift to endosomes and the vacuole ( Figure 9D). ...
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... bar, 5 µm. defects observed during glucose starvation and PI3K-Gpa1 regulation was noted due to a constitutively active Gpa1 Q323L mutant also being defective in recycling ( Figure 5). The screen that discovered that signaling of Gpa1 Q323L requires PI3K also revealed that reg1∆ cells, which lack the phosphatase subunit Reg1 ( Tu and Carlson, 1995;Sanz et al., 2000), gave the largest defect in signaling across all ∼5000 mutants tested (Slessareva et al., 2006). ...
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... support of this hypothesis, we observe recycling defects in both reg1∆ mutants ( Figure 5, E and F) but also in mig1∆ mig2∆ cells ( Figure 7C) lacking downstream transcriptional repressor activity (Schüller, 2003). This suggested that expression of an unknown inhibitor of Gpa1-mediated recycling would be derepressed following glucose starvation or in mig1∆ mig2∆ mutants. ...
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... To quantify 67 this difference, measurements of cellular area segmented from brightfield micrographs showed a mean area of 68 17.2 ± 5.8 µm 2 for S. cerevisiae compared with 10.2 ± 5.8 µm 2 for D. hansenii ( Figure 1B). We have previously 69 noted that this type of analysis from dividing cells results in a wide range of areas (Laidlaw et al., 2022a), so 70 we sampled more than 600 cells per condition. To underscore this difference, we cultured a strain of S. ...
Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and different membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied.
... A similar dual control of trafficking pathways in response to glucose starvation, which triggers surface protein degradation (Lang et al., 2014), occurs through an increase in AP180 mediated endocytosis and a decrease in Gpa1-PI3-kinase mediated recycling Laidlaw et al., 2022b). ...
The yeast plasma membrane (PM) is organised into specific subdomains that regulate surface membrane proteins. Surface transporters actively uptake nutrients in particular regions of the PM where they are also susceptible to substrate induced endocytosis. However, transporters also diffuse into distinct subdomains termed eisosomes, where they are protected from endocytosis. Although most nutrient transporter populations are downregulated in the vacuole following glucose starvation, a small pool is retained in eisosomes to provide efficient recovery from starvation. We find the core eisosome subunit Pil1, a Bin, Amphiphysin and Rvs (BAR) domain protein required for eisosome biogenesis, is phosphorylated primarily by the kinase Pkh2. In response to acute glucose starvation, Pil1 is rapidly dephosphorylated. Enzyme localisation and activity screens implicate the phosphatase Glc7 is the primary enzyme responsible for Pil1 dephosphorylation. Both depletion of GLC7 and phospho-ablative or phospho-mimetic mutations of Pil1 correlate with Pil1 phosphorylation status, failure to properly retain transporters in eisosomes, and results in defective starvation recovery. We propose precise posttranslational control of Pil1 modulates nutrient transporter retention within eisosomes depending on extracellular nutrient levels, to maximise recovery following starvation.
... Surface proteins are also degraded faster and recycled less efficiently in response to leucine starvation (Jones et al., 2012;MacDonald and Piper, 2017). A similar dual control of trafficking pathways in response to glucose starvation, which triggers surface protein degradation (Lang et al., 2014), occurs through an increase in AP180 mediated endocytosis and a decrease in Gpa1-PI3-kinase mediated recycling (Laidlaw et al., 2021;Laidlaw et al., 2022). ...
The yeast plasma membrane (PM) is organised into specific subdomains that can regulate the activity of surface membrane proteins localised within them. Nutrient transporters actively uptake substrates in particular regions of the PM where they are also susceptible to the endocytic machinery for substrate induced degradation. However, transporters also diffuse into distinct subdomains termed eisosomes, where they are inactive for substrate uptake and are protected from endocytosis. Although most nutrient transporter populations are sorted to the vacuole for degradation during glucose starvation, a small pool are retained in eisosomes. Sequestering this small pool of transporters during nutrient stress is essential for efficient recovery from starvation following a return to replete conditions. However, the mechanisms controlling this process at a biochemical level are poorly defined. We find the core eisosome subunit Pil1, a Bin, Amphiphysin and Rvs. (BAR) domain protein involved in membrane dynamics required for eisosome biogenesis, is primarily phosphorylated by Pkh2 but that Pil1 dephosphorylation occurs during acute glucose starvation. We screened for enzyme localisation and activity to implicate the essential phosphatase Glc7 as the primary enzyme responsible for Pil1 dephosphorylation. Manipulation of GLC7 expression correlates with Pil1 hypo/hyper phosphorylation. Furthermore, glc7 mutants and Pil1-phosphomutants are defective in recovering from glucose starvation. We propose precise posttranslational control of Pil1 modulates nutrient transporter retention within eisosomes depending on cellular nutritional status, to maximise recovery from starvation.
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.
Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both “early” and “recycling” endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved.
