Ypt1/Rab1 regulates Hrr25/CK1δ kinase activity in ER-Golgi traffic and macroautophagy

Abstract

ER-derived COPII-coated vesicles are conventionally targeted to the Golgi. However, during cell stress these vesicles also become a membrane source for autophagosomes, distinct organelles that target cellular components for degradation. How the itinerary of COPII vesicles is coordinated on these pathways remains unknown. Phosphorylation of the COPII coat by casein kinase 1 (CK1), Hrr25, contributes to the directional delivery of ER-derived vesicles to the Golgi. CK1 family members are thought to be constitutively active kinases that are regulated through their subcellular localization. Instead, we show here that the Rab GTPase Ypt1/Rab1 binds and activates Hrr25/CK1δ to spatially regulate its kinase activity. Consistent with a role for COPII vesicles and Hrr25 in membrane traffic and autophagosome biogenesis, hrr25 mutants were defective in ER-Golgi traffic and macroautophagy. These studies are likely to serve as a paradigm for how CK1 kinases act in membrane traffic.

Figure 1.

Hrr25 is required for ER–Golgi traffic in vivo and COPII vesicle fusion in vitro. (A) Vesicle budding and (B) fusion were measured in vitro in WT (SFNY2051) and hrr25-5 mutant (SFNY 2049) fractions as described in the Materials and methods. (C) Increasing concentrations of purified recombinant His₆-Hrr25 were incubated with a mutant S1 fraction for 30 min on ice before the transport assay was performed. (A–C) Error bars represent SD; n = 3; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, Student’s t test. Internal (D) and external (E) forms of invertase were examined in WT (SFNY 2330), hrr25-5 (SFNY 2329), and bet2-1 (SFNY 92) mutant cells. The ratio of internal to external invertase is reported at the bottom of each lane. (F) The external form of invertase secreted in WT, hrr25-5, and bet2-1 mutant cells was treated with endoglycosidase H (Endo H).

Figure 2.

Ypt1 regulates the recruitment of Hrr25 to membranes. (A) Cells expressing Ypt1 Q67L (SFNY2520, lane 3) or S22N (SFNY2521, lane 4) were immunoprecipitated from lysates in which Hrr25 was tagged with HA or not tagged (untagged, lanes 1 and 2). The entire precipitate was immunoblotted with anti-Ypt1 antibody. Lanes 5 and 6, input for Ypt1 Q67L. (B) Quantitation of the coimmunoprecipitation of Ypt1 Q67L and S22N with Hrr25-HA. Error bars represent SEM; n = 3; *, P < 0.05, Student’s t test. (C, left) WT (SFNY 445) and the ypt1-3 mutant (SFNY 446) were shifted to 37°C for 1 h. Total lysates (T) were prepared and fractionated into supernatant (S) and pellet (P) fractions. The membrane protein Bos1 and cytoplasmic phosphoglycerate kinase 1 (Pgk1) were used as fractionation controls. (right) Quantitation of differential fractionation experiments. Error bars represent SEM; n = 3; **, P < 0.01 Student’s t test.

Figure 3.

CK1δ is a Rab1 effector. (A) Purified GST or GST-CK1δ was incubated with increasing amounts of purified His₆-Rab1a Q70L (left) or His₆-Rab1a S25N (right). (B) Same as A, except His₆-Rab2a, His₆-Rab1a, and His₆-Rab4a were incubated with GST and GST-CK1δ (C) HeLa cells were harvested 96 h after they were mock transfected or transfected with the shRab1a construct. Samples were immunoblotted with anti-Rab1 and anti-actin antibodies. Actin was used as a loading control. In three separate experiments, ∼90.1 ± 0.4% of the Rab1a was depleted in the shRNA-treated cells. (D) Lysates (T) were prepared from mock and depleted cells and fractionated as previously described (Bhandari et al., 2013) to produce supernatant (S) and pellet (P) fractions. Western blot analysis was performed to detect CK1δ, calnexin, and the cytoplasmic protein GAPDH in each fraction. (E) Quantitation of differential fractionation. Error bars represent SEM; n = 4; *, P < 0.05, Student’s t test.

Figure 4.

Ypt1 regulates Hrr25 kinase activity on vesicles. (A) Hrr25 kinase activity is ts in the ypt1-3 mutant. WT (SFNY 2443) and mutant (SFNY 2445) cells were grown at 23°C or shifted to 37°C for 2 h. Hrr25-HA was precipitated from lysates onto Protein A–conjugated agarose beads and kinase activity was assayed using MBP as a substrate as described in the Materials and methods. (B) Quantitation of kinase activity from WT and the ypt1-3 mutant from three separate experiments. The data were normalized to the amount of Hrr25-HA in the precipitate. Error bars represent SD; n = 3; *, P < 0.05, Student’s t test. (C and D) Hrr25-HA was precipitated from permissively grown ypt1-3 cells and preincubated with increasing concentrations of Ypt1 Q67L (C) or Ypt1 (D) for 15 min at 25°C and then assayed as described in the Materials and methods. The ratio of phosphorylated MBP to Hrr25-HA is calculated at the bottom of each lane. Note that the kinase activity of the mutant at 0 ng was set at 1.0. The assays in C and D were performed multiple times. The data that are shown are representative. (E) Lst1 is hypophosphorylated in the ypt1-3 and sec12-4 (NY738) mutants. WT and mutant cells were shifted to 37°C for 1 h, lysates were prepared and Lst1 was immunoprecipitated (lanes 1–12). Samples in lanes 5, 6, 9, and 10 were treated with CIP, while samples in lanes 7, 8, 11, and 12 were treated with CIP plus EDTA.

Figure 5.

Ypt1 recruits Hrr25 to the macroautophagy pathway. (A) Hrr25-GFP appears to be more cytosolic in the ypt1-2 (SFNY2528) and sec12-4 (SFNY2569) mutants than WT. For the ypt1-2 mutant, the WT strain was SFNY2527. For the sec12-4 mutant, the WT strain was SFNY 2568. Cells expressing Hrr25-GFP and Ape1–RFP were grown to log phase in SC–Leu medium at 25°C and shifted to SD–N medium for 4 h at 25°C. The sec12-4 mutant and the corresponding WT were shifted to SD–N medium for 2 h at 37°C. Arrowheads point to the PAS, which is marked with Ape1-RFP. Bar, 2 µm. (B) The ApeI-RFP that colocalized with Hrr25-GFP was calculated in 450 cells. Error bars represent SEM; n = 3; *, P < 0.05; Student’s t test.

Figure 6.

The COPII coat is required for macroautophagy, but not the Cvt pathway. (A) Vacuolar alkaline phosphatase activity was assayed in SFNY2529, SFNY2530, SFNY2565, and SFNY2567 as described in the Materials and methods. The activity of WT after 2 h of starvation at 37°C was set as 100% and the activity at time 0 was subtracted. Error bars represent SEM; n = 3; *, P < 0.05; **, P < 0.01, Student’s t test. (B, left) The translocation of GFP-Atg8 was examined in WT (SFNY 2623), hrr25-5 (SFNY 2622), ypt1-2 (SFNY 2660), and ypt1-3 (SFNY 2662) mutants 2 h after nitrogen starvation at 37°C. Bar, 2 µm. (right) Error bars represent SEM; n = 3; **, P < 0.01, Student’s t test. (C) Vacuolar alkaline phosphatase was measured in protein extracts of WT (SFNY2554), the sec23 phosphomimetic (SFNY 2556), and alanine (SFNY 2555) mutants. Error bars represent SEM; n = 3; *, P < 0.05 Student’s t test. (D) The translocation of GFP-Atg8 was examined in WT (SFNY2624) and the sec23 mutants (SFNY2625, SFNY2626) 1 h after nitrogen starvation at 37°C. Error bars represent SEM; n = 3; *, P < 0.05, Student’s t test. Bar, 2 µm. (E and F) Hrr25, but not Sec23, is required on the Cvt pathway (SFNY1950, SFNY1951, SFNY1952, and SFNY2488). Ape1 processing was measured as described in the Materials and methods. (G) WT, atg1Δ, and atg13Δ cells expressing Sec13-GFP and Ape1-RFP (SFNY2627, SFNY2628, and SFNY2629) were grown to log phase in SC–Leu medium at 25°C. Cells from three separate experiments (450 total) were examined to calculate the percentage of Ape1-RFP puncta that colocalize with (left) or lie adjacent to (right) Sec13-GFP puncta.

Figure 7.

Hrr25 and Ypt1 are required for autophagosome formation. (A) WT cells expressing GFP-Atg8 were grown to log phase and treated with 400 ng/ml rapamycin for 1 h at 25°C. Deconvolved images are shown. Bars, 1 µm. (B) WT (SFNY 2663) and hrr25Δ (SFNY 2664) cells expressing GFP-Atg8 were treated with 400 ng/ml rapamycin for 30 min at 25°C. The ypt1-3 (SFNY2662) and hrr25-5 (SFNY 2622) mutants were treated with rapamycin for 1 h at 37°C. Numbers of autophagosomes were calculated in 300 cells. Error bars represent SEM; n = 3; *, P < 0.05; **, P ≤ 0.01; Student’s t test. (C) WT, hrr25Δ, ypt1-3, and hrr25-5 mutant cells expressing GFP-Atg8 and Ape1-RFP (SFNY2668, SFNY2669, SFNY2670, SFNY2671, and SFNY2696) were treated as above. Arrowheads point to the PAS. Bar, 2 µm. Error bars represent SEM; n = 3; *, P < 0.05; **, P < 0.01, Student’s t test.