A Snf1-related nutrient-responsive kinase antagonizes endocytosis in yeast

Abstract

Endocytosis is regulated in response to changing environmental conditions to adjust plasma membrane (PM) protein composition for optimal cell growth. Protein networks involved in cargo capture and sorting, membrane sculpting and deformation, and vesicle scission have been well-characterized, but less is known about the networks that sense extracellular cues and relay signals to trigger endocytosis of specific cargo. Hal4 and Hal5 are yeast Snf1-related kinases that were previously reported to regulate nutrient transporter stability by an unknown mechanism. Here we demonstrate that loss of Hal4 and Hal5 activates endocytosis of many different kinds of PM proteins, including Art1-mediated and Art1-independent endocytic events. Acute inhibition of Hal5 in the absence of Hal4 triggers rapid endocytosis, suggesting that Hal kinases function in a nutrient-sensing relay upstream of the endocytic response. Interestingly, Hal5 localizes to the PM, but shifts away from the cell surface in response to stimulation with specific nutrients. We propose that Hal5 functions as a nutrient-responsive regulator of PM protein stability, antagonizing endocytosis and promoting stability of endocytic cargos at the PM in nutrient-limiting conditions.

Fig 1. Hal4 and Hal5 cluster in a family of kinases that includes Snf1.

A multiple sequence alignment of all 130 known protein kinases in yeast was performed using Clustal Omega and visualized as a scaled, rooted phylogenetic tree using iTOL. The protein kinases cluster into 6 major clades, which have been arbitrarily numbered and color-coded for simplicity and ease of viewing across different figures. A heat-map to the right of the phylogenetic tree conveys the number of publications annotated in SGD per kinase. Dots to the right of the heat map indicate the classical family assignment for each kinase.

Fig 2. Kinase clustering with Snf1 is driven primarily by catalytic domain similarity.

A multiple sequence alignment of the kinases that clustered with Snf1 in yeast was performed using Clustal Omega and visualized as a scaled, rooted phylogenetic tree using EvolView. To the right of the phylogenetic tree, sequence homology for each kinase with Snf1 is displayed as a 4-column heat-map divided into two sections. In the first section labeled BLAST, percent identity (%ID) or percent similarity (%SIM) was calculated by aligning the two sequences using NCBI-BLAST. In the second section labeled SMS percent identity (%ID) or percent similarity (%SIM) was calculated by aligning the two sequences using Sequence Manipulation Suite [107]. Both programs were used due to having different methods for calculating homology. BLAST calculates homology based on only the aligned region, which in every case is restricted to mostly the catalytic domains. While SMS also aligns sequences, its calculation accounts for the entirety of the protein sequences, demonstrating lower sequence homology outside of the catalytic domains. To the right of the sequence homology heat-map are protein architecture maps for each kinase drawn to scale. These were automatically generated in EvolView from data available for each kinase in UniProt. YPL150W is not annotated in UniProt, and therefore does not have a protein architecture map.

Fig 3. Hal kinases contribute to regulation of Art1-mediated and Art1-independent endocytosis.

(A) Representative images of Mup1-GFP expressed from a centromeric plasmid under native promoter control in the presence of endogenously MARS tagged Vph1, a marker for the limiting membrane of the vacuole. WT, Δhal4Δhal5 cells, or Δhal4Δhal5Δart1 cells were cultured to mid-log phase in selective media and treated with LatA for 1 hour where indicated. (B) Quantification of Mup1-GFP localization in (A) performed by binning cells into localization categories as indicated. (C) Representative images of Fur4-GFP under conditions previously described in (A). (D) Quantification of Fur4-GFP localization in (C) performed as described in (B). (E) Representative images of Pma1-GFP under conditions previously described in (A). (F) Quantification of Pma1-GFP localization in (E) performed as described in (B). (G) Representative images of Pma2-GFP under conditions previously described in (A).

Fig 4. Hal kinases regulate the PM stability of the cell wall integrity sensor Wsc1.

(A) Representative images of Wsc1-GFP expressed from a centromeric plasmid under native promoter control in the presence of endogenously MARS tagged Vph1, a marker for the limiting membrane of the vacuole. WT, Δhal4Δhal5 cells, or Δhal4Δhal5Δart1 cells were cultured to mid-log phase in selective media. (B) Quantification of Wsc1-GFP localization in (A) performed by binning cells into localization categories as indicated. (C) Representative images of a GFP-tagged Wsc1^NPF→AAA variant under conditions previously described in (A). (D) Quantification of Wsc1^NPF→AAA localization in (C) performed as described in (B). (E) Representative images of a GFP-tagged Wsc1^6K→R variant under conditions previously described in (A). (F) Quantification of Wsc1^6K→R localization in (E) performed as described in (B). VM indicates vacuole membrane localization.

Fig 5. Hal4 and Hal5 exhibit redundant roles with respect to nutrient transporter localization.

(A) Representative images of Mup1-GFP expressed from a centromeric plasmid under native promoter control in the presence of endogenously MARS tagged Vph1, a marker for the limiting membrane of the vacuole. WT, Δhal4 or Δhal5 single mutant cells or hal double mutant cells were cultured to mid-log phase in selective media. (B) Quantification of Mup1-GFP localization in (A) performed by counting a population of cells and binning each cell into a cargo localization category (PM only, PM + Vac, or Vac only). (C) Percentage of cell population expressing endogenously tagged Mup1-pHluorin as measured by cells that fall within a defined FITC gate (green fluorescence) by flow cytometry at steady state (10,000 cells counted per condition, n = 4 biological replicates). (D) Percentage of cell population expressing endogenously tagged Mup1-pHluorin as measured by cells that fall within a defined FITC gate (green fluorescence) by flow cytometry (10,000 cells counted per condition, n = 4 biological replicates) over time in the presence of excess methionine, an endocytic stimulant. Mup1-pH PM half-time (t_1/2) was estimated based on initial and final time points and elapsed time. (E) Representative images of Fur4-GFP under conditions previously described in (A). (F) Quantification of Fur4-GFP localization in (E) performed as described in (B).

Fig 6. Hal5 catalytic activity antagonizes the endocytic trafficking of Mup1.

(A) Schematic representation of Hal5 sequence alignment with Snf1 to identify Met620 as the gatekeeper residue. Snf1 is the most-related kinase for which an analog-sensitive allele has been previously characterized (snf1AS: I132G inhibited by 2-NM-PP1) (B) Cells were serially diluted onto synthetic selective media and grown for 3 days to assay functionality of hal5AS by growth. (C) Representative image of Δhal4,5 mutant cells expressing either WT (HAL5-HTF) or analog-sensitive (hal5AS-HTF) Hal5 spread onto synthetic selective media as a lawn. Whatman paper disks soaked in a solution of 1-NA-PP1 dissolved in vehicle (EtOH) at the indicated concentrations were placed on top of the lawn prior to incubation to establish a concentration gradient of inhibitor. Plates were grown for 3 days to assess cell growth, and therefore inhibition of hal5AS by 1-NA-PP1 (structure shown in right of panel). (D) Representative images of Mup1-GFP localization in Δhal4,5 mutant cells exogenously expressing either HAL5 (white arrow indicators) or hal5AS (magenta arrow indicators, cells marked by Vph1-MARS, a marker of the vacuolar limiting membrane). Cells were co-cultured in synthetic selective media to mid-log phase then treated with vehicle (EtOH) or inhibitor (1-NA-PP1 26.3μM) for 1 hr, then imaged. (E) Immunoblot analysis of Δhal4,5 mutant cells exogenously expressing empty vector (ev), WT (HAL5-HTF), or analog-sensitive (hal5AS-HTF) Hal5, under indicated conditions to asses Hal5 protein expression. (F) Percentage of cell population expressing endogenously tagged Mup1-pHluorin as measured by cells that fall within a defined FITC gate (green fluorescence) by flow cytometry at steady state (10,000 cells counted per condition, n = 3 biological replicates) over time in the presence of hal5AS inhibitor 1-NA-PP1 or mock treatment (EtOH). WT or analog-sensitive HAL5 (HAL5-HTF or hal5AS-HTF) is exogenously expressed in Δhal4,5 or 5 mutant cells from a centromeric plasmid under native promoter control. EV indicates empty vector.

Fig 7. Acute inhibition of Hal5 activity triggers rapid endocytic clearance of nutrient transporters.

(A) Representative images of Mup1-GFP expressed from a centromeric plasmid in the presence of endogenously-tagged Vph1-MARS, a marker for the limiting membrane of the vacuole. WT or analog-sensitive HAL5 (HAL5-HTF or hal5AS-HTF) is exogenously expressed in Δhal4,5 or Δhal4,5Δart1 mutant cells from a centromeric plasmid under native promoter control as indicated. Cells were grown to mid-log phase in selective media and imaged after 1 hr of inhibitor treatment (1-NA-PP1). Where indicated, cells were pre-treated with LatA for 1 hr prior to 1-NA-PP1 inhibition. (B) Quantification of Mup1-GFP localization in (A) performed by binning cells into localization categories as indicated. (C) Percentage of cell population expressing endogenously tagged Mup1-pHluorin as measured by cells that fall within a defined FITC gate (green fluorescence) by flow cytometry at steady state (10,000 cells counted per condition, n = 3 biological replicates) over time in the presence of hal5AS inhibitor 1-NA-PP1. WT or analog-sensitive HAL5 (HAL5-HTF or hal5AS-HTF) is exogenously expressed in Δart1Δhal4,5 mutant cells from a centromeric plasmid under native promoter control. EV indicates empty vector. (D) Representative images of Fur4-GFP under conditions described previously in (A). (E) Quantification of Fur4-GFP localization in (D) performed as described in (B).

Fig 8. N-terminal elements of Hal5 are critical for antagonizing nutrient transporter endocytosis.

(A) Schematic representation of Hal5 truncation variants compared to WT Hal5. (B) Representative image of cells serially diluted on synthetic selective media and grown for 3 days. (C) Immunoblot analysis to examine expression of Hal5 variants that fail to complement growth in hal mutant cells. (D) Representative images of Mup1-GFP expressed from a centromeric plasmid in the presence of endogenously-tagged Vph1-MARS, a marker for the limiting membrane of the vacuole. Hal5 variants are exogenously expressed in Δhal4,5 mutant cells from a centromeric plasmid under native promoter control. (E) Quantification of Mup1-localization in (D) performed by binning cells into localization categories as indicated. (F) Percentage of cell population expressing endogenously tagged Mup1-pHluorin as measured by cells that fall within a defined FITC gate (green fluorescence) by flow cytometry at steady state (10,000 cells counted per condition, n = 3 biological replicates).

Fig 9. N-terminal elements of Hal5 are critical for localization to the PM.

(A) Representative image of WT cells grown to mid-log phase in selective media expressing Hal5 C-terminally-tagged with mNeonGreen (Hal5-mNG) from a centromeric plasmid under native promoter control. (B) Schematic representation of C-terminally mNeonGreen-tagged Hal5 variants compared to WT Hal5. (C) Representative images of WT cells grown to mid-log phase in selective media expressing variants of Hal5-mNG after a brief FM 4–64 pulse to label PM immediately prior to imaging (D) Hal5 localization to the PM was quantified in (C) by measuring Pearson correlation coefficient of Hal5-mNG signal with FM 4–64 signal. Standard deviation of cells expressing full-length Hal5-mNG is denoted by the green box. (E) Table summarizing each Hal5 variant tested, its functionality, and its localization.

Fig 10. Nutrient availibility regulates Hal5 localization.

(A) Representative images of WT cells expressing full-length Hal5-mNG from a centromeric plasmid under native promoter control. Cells were grown to mid-log phase in selective media then switched to media with the indicated nutrient conditions (10 μg/mL methionine (+ MET), or 10 μg/mL uracil (+ URA)) for 10 minutes, then briefly pulsed with FM 4–64 to label PM immediately prior to imaging. (B) Hal5 localization to the PM was quantified in (A) by measuring Pearson correlation coefficient of Hal5-mNG signal with FM 4–64 signal. Standard deviation of control cells expressing full-length Hal5-mNG is denoted by the green box.