Abstract
COPII coated vesicles that bud from the endoplasmic reticulum (ER) normally traffic to the Golgi. However, during starvation, COPII vesicles are re-directed to the macroautophagy pathway where they become a membrane source for autophagosomes. Phosphorylation of the coat by the casein kinase 1 (CK1), Hrr25, is a prerequisite for vesicle uncoating and membrane fusion. CK1 family members were initially thought to be constitutively active kinases that are regulated through their subcellular localization. Recent studies, however, have shown that the Rab GTPase Ypt1 binds to and activates Hrr25 (CK1δ in mammals) to spatially regulate its kinase activity. Consistent with a direct role for Hrr25 in macroautophagy, hrr25 and ypt1 mutants are defective in autophagosome biogenesis. These studies have provided insights into how the itinerary of COPII vesicles is coordinated on two different trafficking pathways.
Keywords: Rab GTPase, Ypt1, casein kinase, Hrr25, vesicle traffic
1. Introduction
ER-Golgi transport vesicles bud from the ER upon the hierarchical assembly of the COPII coat [1, 2]. The COPII coat is formed when the activated form of the GTPase Sar1 (Sar1-GTP) recruits the cargo adaptor complex, Sec23-Sec24, that sorts cargo into the vesicle. Sec23-Sec24 then recruits the coat outer shell, Sec13-Sec31 [1,2]. After cargo packaging and vesicle scission, COPII vesicles are transported to the Golgi. The directional delivery of ER-derived vesicles to the Golgi is controlled by the sequential interactions of the COPII coat subunit Sec23 and three of its binding partners: Sar1-GTP, TRAPPI and Hrr25[3]. TRAPPI is aguanine nucleotide exchange factor (GEF) that activates the GTPase Ypt1[4], and Hrr25 is a CK1 kinase family member [5]. The fidelity of vesicle traffic requires Rab GTPases and their effectors [6]. There are 11 Rabs in yeast (called Ypt) and over 60 Rabs in mammalian cells that regulate a variety of membrane trafficking steps [7]. Ypt1 and its mammalian homolog Rab1 function in ER to Golgi traffic, intra-Golgi traffic and macroautophagy (herein called autophagy) [4,8]. The phosphorylation of multiple COPII coat subunits by Hrr25 is a prerequisite for vesicle uncoating and the pairing of the fusion machinery (SNAREs) on the vesicle and target membrane (Figure 1) [3,9].
Figure 1. Hrr25 kinase activity is regulated by the Rab GTPase Ypt1.
(1) After Sar1 is released from the vesicle, TRAPPI binds to Sec23 and activates (2) Ypt1. (3) Ypt1-GTP then recruits its effectors Hrr25 and the tether (Uso1) that links the vesicle to the Golgi. (4) Activated Hrr25 then phosphorylates the vesicle-bound coat.
When autophagy is induced, COPII vesicles are diverted to the pre-autophagosomal structure (PAS) where they contribute membrane for autophagosome biogenesis[10,11]. Hrr25 phosphorylates the membrane distal surface of Sec24 which enables this COPII coat subunit to bind to Atg9, a transmembrane protein required for autophagosome initiation [12].
Here we present the methods used in Wang et al. (2015) that address how Hrr25 specifically phosphorylates the vesicle–borne pool of the COPII coat on two different pathways: ER-Golgi and autophagy [13]. These methods also address how Wang et al. (2015) showed that Hrr25 is an effector of Ypt1[13]. Ypt1 directly recruits Hrr25 to COPII vesicles to activate it. This was the first report that the activity of a CK1 family member is regulated by a Rab GTPase.
2. Materials
2.1. Culture media
1. You should use several different types of growth medium for these studies. For growth in rich medium, (YPD): 1% yeast extract, 2% peptone, and 2% dextrose or synthetic minimal media (SMD): 0.67% yeast nitrogen base, 2% dextrose and auxotrophic amino acids as needed. For solid media, agar is added to a final concentration of 2%.
2. You should induce autophagy by starving cells in synthetic minimal medium lacking nitrogen (SD-N)): 0.17% yeast nitrogen base without amino acids and ammonium sulfate, and 2% dextrose.
2.2. Solutions
1. Bacterial lysis buffer 1: 20 mM Tris pH 8, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, and 15 mM Imidazole
2. Bacterial lysis buffer 2: 1x PBS, 1 mM PMSF and 1 mM DTT
3. Elution buffer: 20 mM Tris pH 8, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, and 250 mM Imidazole
4. Binding buffer: 50 mM HEPES pH 7.2, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 2% Triton X100, and 5 x protease inhibitor cocktail (PIC) from Roche
5. SDS sample buffer: 50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, and 0.02 % bromophenol blue
6. Yeast lysis buffer 1: 1x PBS, 200 mM sorbitol, 1 mM MgCl2, 0.1% Tween 20, and PIC
7. Spheroplast buffer:1.4 M sorbitol, 10 mM Na azide, 50 mM KPi, pH 7.5, 0.35% β-mercaptoethanol, and 1 mg zymolase
8. Sorbitol cushion: 1.7 M sorbitol, and 100 mM HEPES, pH 7.4
9. Yeast lysis buffer 2: 100 mM HEPES, pH 7.2, 1 mM EGTA, 0.2 mM DTT, 1 mM PMSF, and PIC
10. Tris-HCl buffer: 50 mM Tris-HCl pH 7.4, 100 mM NaCl.
11. Immunoprecipitation buffer: 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 1 mM PMSF, and PIC
12. Mammalian lysis buffer: 100 mM HEPES buffer pH 7.2, 1 mM EDTA, 0.2 mM DTT, 5 x PIC, 5 x phosphatase inhibitor cocktail
13. Kinase buffer: 50 mM HEPES, pH 7.4, 5 mM MgCl2, 0.2% Nonidet P-40, and 1 mM DTT
2.3. Reagents
1. Ni-NTA Agarose
2. Glutathione-sepharose beads
3. Spin column with a cut-off filter of 10 kDa
4. Nitrocellulose membranes
5. Anti-His antibody
6. Anti-HA antibody
7. Anti-rabbit IgG
8. Anti-mouse IgG
9. HA affinity matrix
10. Enhanced chemiluminescence (ECL) detection kit
11. QuickChange site-directed mutagenesis kit (Stratagene).
12. shRNA expression vector pSilencer 1.0-U6 (Ambion).
13. Transfection reagents: OPTI-MEM (Invitrogen) and Lipofectamine 2000 (Life Technologies).
3. Methods
3.1. An in vitro binding assay for Rab1A and CK1δ (mammalian homologue of Hrr25)
To address if CK1δ is an effector of Rab1A, you should test the interaction between CK1δ and Rab1A (GTP or GDP-locked form). Purified recombinant GST-CK1δ is incubated with His6-Rab1A Q70L (GTP- locked form) and His6-Rab1A S25N (GDP- locked form). GST-CK1δ should bind to Rab1A Q70L, but not Rab1A S25N.
3.1.1. Purification of recombinant His6-Rab1A Q70L and His6-Rab1A S25N.
1. You can use human Rab1A cloned into the pET-15b vector. Q70L and S25N point mutations are made using the QuickChange site-directed mutagenesis kit. His6-Rab1A expressing plasmids are transformed into Rosetta (DE3) cells.
2. A single colony is inoculated in 50 ml of LB medium with 100 μg/ml ampicillin and grown overnight at 37 °C.
3. An overnight culture is inoculated into 1 L of LB medium containing 100 μg/ml ampicillin to a starting OD600 of approximately 0.1 and grown at 37°C. When the OD600 reaches 0.6, filter sterilized IPTG is added to a final concentration of 0.5 mM. His6-Rab1A Q70L and His6-Rab1A S25N expression is induced overnight at 20°C.
4. Cells are centrifuged at 6000 x g for 5 min and resuspended in 20 ml of bacterial lysis buffer 1.
5. The cells are sonicated eight times for 15 sec intervals at 50% amplitude.
6. The sample is centrifuged at 12, 000 x g for 15 min at 4 °C.
7. The cleared lysate is transferred into a fresh 50 ml tube. Prewashed 2 ml Ni-NTA agarose beads are added and incubated at 4 °C with rotation (20 rpm) for 30 min (see Note 1).
8. The slurry of Ni-NTA beads and lysate is loaded onto a 0.8 x 12 cm polypropylene column and washed with 20 ml of bacterial lysis buffer 1.
9. The bound protein is eluted with 8 ml of elution buffer.
10. A spin column, containing a cut-off filter of 10 kDa, is used to concentrate the eluate to approximately 0.5 ml.
11. Purified His6-Rab1A is analyzed on a 13% SDS-PAGE gel and stained with Coomassie Brilliant Blue. The protein concentration is measured using BSA as a standard and analyzed using AlphaView software.
3.1.2. Purification of glutathione-sepharose beads containing GST and GST fusion proteins.
1. A single colony is inoculated in 50 ml of LB medium with 100 μg/ml ampicillin and incubated overnight at 37 °C.
2. An overnight culture is inoculated into 1 L of LB medium with 100 μg/ml ampicillin at 37 °C with a starting OD600 of approximately 0.1. When the OD600 reaches 0.6, IPTG is added to a final concentration of 0.5 mM to induce the expression of GST and GST fusion proteins during an overnight incubation at 20 °C.
3. Cells are centrifuged at 6000 x g for 10 min and resuspended in 20 ml of bacterial lysis buffer 2.
4. The suspension is sonicated at 50% amplitude for eight times with 15 sec intervals.
5. Triton X-100 is added to the sample to a final concentration of 1%.
6. The sample is centrifuged at 12, 000 x g for 15 min.
7. The cleared lysate is transferred into a fresh 50 ml tube. Prewashed glutathione-sepharose beads (1 ml) are added and incubated at 4 °C with rotation (20 rpm) for 30 min (see Note 2).
8. The beads are centrifuged at 500 x g for 2 min and washed three times with 10 ml of PBS.
9. SDS-PAGE sample buffer (50 μl) is added to the glutathione-sepharose beads. The bound proteins are analyzed on an 8% SDS-PAGE gel and stained with Coomassie Brilliant Blue. The protein concentration of GST and GST fusion proteins are measured using BSA as a standard and analyzed using AlphaView software.
3.1.3. In vitro binding assay with GST- CK1δ and His6-Rab1A (Q70L and S25N).
1. For these studies, you can clone human CK1δ into plasmid pGEX-4T-2. The plasmid is transformed into BL21(DE3) cells to express GST-CK1δ. Varying concentrations of His6-Rab1A (Q70L or S25N) (examples include 125 nM, 250 nM, 500 nM, 1000 nM) are incubated with equimolar amounts (0.1μM) of immobilized GST or GST-CK1δ in a total volume of 500 μl of binding buffer for 3 hr at 4 °C with rotation (20 rpm) (see Note 3 and 4).
2. The beads are washed three times with 1ml of binding buffer without protease inhibitors.
3. The beads are resuspended in 50 μl of SDS-PAGE sample buffer and heated at 100 °C for 5 min.
4. The eluate is fractionated on a 13% SDS-PAGE gel and transferred onto nitrocellulose membranes in transfer buffer.
5. The membranes are blocked with blocking solution for 1 hr.
6. Anti-His antibody is added to the membranes and incubated at 4 °C overnight.
7. The membranes are washed three times with 10 ml of the TBST buffer.
8. Anti-mouse IgG (1:10000 dilution) is added and incubated for 1hr.
9. The membranes are washed three times with 10 ml of TBST buffer and then analyzed using the ECL method.
3.2. Differential centrifugation can be used to assess if Ypt1 and Rab1 regulate the recruitment of Hrr25 and CK1δ to membranes.
You can address the effect of Ypt1 on the membrane distribution of Hrr25 in yeast in vivo. The membrane distribution of Hrr25 can be examined in the temperature-sensitive ypt1-3 mutant. In mammalian cells, the distribution of CK1δ can be monitored in Rab1A knockdown HeLa cells. WT and ypt1-3 mutant yeast cells are incubated for 1hr at 37 °C before cell lysates are fractionated by differential centrifugation into soluble and insoluble fractions. A significant increase in the soluble pool of Hrr25 can be observed in the ypt1-3 mutant. A reproducible increase in the soluble pool of CK1δ can also be seen in Rab1A depleted HeLa cells.
3.2.1. Examining the membrane distribution of Hrr25 in WT and the ypt1-3 mutant.
1. WT and ypt1-3 cells are grown in YPD at 25 °C and then shifted to 37 °C for 1 hr.
2. Eighty OD600 units of cells are harvested at 3,000 rpm for 3 min and converted to spheroplasts in 2 ml of spheroplast buffer.
3. Spheroplasts are formed during a 45 min incubation at 37 °C, then spun at 3,000 rpm for 5 min through a 2 ml sorbitol cushion.
4. The pellet is resuspended in 1 ml of yeast lysis buffer 2 and lysed with a Dounce homogenizer.
5. The lysate is spun at 500 x g for 2 min and the supernatant (100 μl) is mixed with 50 μl of 3 x sample buffer (total fraction) and heated to 100 °C for 5 min.
6. The remaining portion (500 μl) is centrifuged at 190,000 x g for 90 min at 4° C. The lipid layer is removed and the supernatant (100 μl) is mixed with 50 μl of 3 x sample buffer (supernatant fraction) and heated to 100 °C for 5 min.
7. The pellet is resuspended in 500 μl of lysis buffer. Then, 100 μl is mixed with 50 μl of 3 x sample buffer and heated to 100 °C for 5 min (pellet fraction).
8.Western blot analysis is performed to detect Hrr25 in total lysates, supernatant and pellet fractions. Membrane and cytoplasmic proteins should be used to monitor the fractionation. The membrane protein Bos1 and cytoplasmic phosphoglycerate kinase 1 (Pgk1) are useful markers for this purpose in budding yeast.
3.2.2. The distribution of CK1δ in HeLa cells after Rab1A depletion.
Rab1A is efficiently knocked down (90%) in HeLa cells using shRab1A. The membrane distribution of CK1δ is analyzed using two different loading controls, a marker for the ER (Calnexin) and a cytoplasmic protein (GAPDH).
1. The shRab1A plasmid used to target human Rab1A, 392–410 (5′-AGAAAGTAGTAGACTACAC-3′) is constructed in the shRNA expression vector pSilencer 1.0-U6.
2. HeLa cells (1.8 x 105/well) are seeded in 6-well plates 20 hr before transfection. The cells are then transfected for 5 hr in OPTI-MEM with 2 μg DNA (pSilencer 1.0-U6 empty vector control or the shRab1a construct) in the presence of 5 μl Lipofectamine 2000 Transfection Reagent.
3. Four days after transfection, the cells are harvested, and washed twice with cold PBS.
4. The cells are lysed in cold mammalian lysis buffer and passed through a 25G needle 10 times using a syringe.
5. The lysate is spun at 500 x g for 2 min and the supernatant (100 μl) is mixed with 50 μl of 3 x sample buffer (total fraction) and heated to 100 °C for 5 min.
6. The remaining portion is centrifuged at 150,000 x g for 90 min at 4 °C. The lipid layer is removed and the supernatant (100 μl) is mixed with 50 μl of 3 x sample buffer (supernatant fraction) and heated to 100 °C for 5 min.
7. The pellet is resuspended in 500 μl of lysis buffer and 100 μl is mixed with 50 μl of 3 x sample buffer and heated to 100 °C for 5 min (pellet fraction).
8. Western blot analysis is performed to detect CK1δ in the lysate, supernatant and pellet fractions.
3.3. Kinase assays
To ask if Ypt1 regulates Hrr25 kinase activity in yeast, Hrr25-HA can be immunoprecipitated from WT and ypt1-3 mutant cells, and kinase activity is measured in vitro using myelin basic protein (MBP) as a substrate. Although equal amounts of Hrr25-HA are precipitated with anti-HA antibody from both strains, the kinase activity of ypt1-3 is significantly lower compared to that of WT.
3.3.1. Immunoprecipitation of Hrr25-HA from a yeast lysate
1. Wild type and ypt1-3 cells expressing Hrr25-HA are grown overnight in SC-URA medium to early log phase and shifted to 37 °C for 2 hr. Cells are collected by centrifugation at 500 x g for 5 min and washed once with Tris-HCl buffer.
2. The washed cells are lysed in 0.5 ml of immunoprecipitation buffer and vortexed with glass beads.
3. The lysates are transferred to fresh tubes, and the remaining glass beads are washed twice with 0.5 ml of lysis buffer.
4. The washes and lysates are combined and Triton X-100 is added to the samples to a final concentration of 0.75%. The samples are incubated at 4 °C on a nutator for 15 min and centrifuged at 10,000 x g for 10 min.
5. Clarified lysates (2 mg) are incubated with 2 μg of anti-HA antibody (12CA5) for 2 hr at 4 °C.
6. Protein-A–conjugated agarose beads (20 μl) are added to the lysates and incubated for an additional 1.5 h (see Note 5).
7. The beads are washed twice with lysis buffer containing 0.75% of Triton X-100 and twice with kinase buffer.
3.3.2. Kinase activity assay
1. The purified Hrr25-HA beads (20 μl) are incubated at 30 °C for 30 min with 2 μg of MBP, 100 μM ATP, and 5 μCi of 32P γATP in 50 μl of kinase buffer.
2. The reaction is terminated by the addition of 20 μl of 4 x SDS sample buffer during a 5 min incubation at 95 °C.
3. The reaction mixture is subjected to SDS/PAGE. The incorporation of radiolabeled 32P into MBP is determined by radioautography and the amount of immunopurified Hrr25-HA is determined by immunoblotting.
3.3.3. Purification of His6-tagged Ypt1 Q67L and WT.
1. Ypt1 is cloned into the pET-29a plasmid and transformed into BL21(DE3) cells to express Ypt1-His6. The Q67L point mutation can be made using the QuickChange site-directed mutagenesis kit. His6-tagged Ypt1 (Q67L and WT) expressing plasmids are transformed into Rosetta (DE3) cells.
2. A single colony is inoculated into 50 ml of LB medium with 30 μg/ml kanamycin and incubated overnight at 37 °C.
3. An overnight culture is inoculated into 1 liter of LB medium containing 30 μg/ml kanamycin at 37 °C with a starting OD600 of approximately 0.1. When the OD600 reached 0.6, filter sterilized IPTG is added to a final concentration of 0.5 mM. Ypt1-His6 expression is induced overnight at 20 °C.
4. Cells are centrifuged at 6000 x g for 5 min and resuspended in 20 ml of bacterial lysis buffer 1.
5. The cells are sonicated for 15 sec at 50% amplitude for a total of 2 min.
6. The samples are centrifuged at 12, 000 x g for 15 min at 4 °C.
7. The cleared lysate is transferred into a fresh 50 ml tube. Prewashed 2 ml Ni-NTA agarose beads are added and incubated at 4 °C with rotation (20 rpm) for 30 min (see Note 3).
8. The slurry of Ni-NTA beads and lysate is loaded onto a 0.8 x 12 cm polypropylene column and washed with 20 ml of bacterial lysis buffer 1.
9. The bound protein is eluted with 8 ml of elution buffer.
10. A spin column containing a cut-off filter of 10 kDa is used to concentrate the eluate to approximately 0.5 ml.
11. Purified Ypt1-His6 is analyzed on a 13% SDS-PAGE gel and stained with Coomassie Brilliant Blue. The protein concentration of Ypt1-His6 is measured using BSA as a standard and analyzed with Alphaview software.
3.3.4. Reconstitution assays
1. The purified Hrr25-HA beads are preincubated with purified Ypt1-His6 (Q67L or wild type) in kinase buffer for 15 min at 25 °C.
2. The purified Hrr25-HA beads and purified Ypt1-His6 (Q67L or wild type) protein are incubated at 30 °C for 30 min with 2 μg of MBP, 100 μM ATP, and 5 μCi of 32P γATP in 50 μl of kinase buffer.
3. The reaction is terminated by the addition of 20 μl of 4 x SDS sample buffer and incubated at 95 °C for 5 min. The reaction mixture is then subjected to SDS/PAGE.
4. 32P labeled MBP is determined by radioautography and the amount of immunopurified Hrr25-HA is determined by immunoblotting.
3.4. The analysis of autophagosome formation by Structured Illumination Microscopy (SIM).
Structured illumination microscopy can be used to visualize the biogenesis of autophagosomes, marked by GFP-Atg8, in deconvolved images of WT, hrr25-5, and ypt1-3 mutant cells (see Note 6).
1. Wild type, hrr25-5 and ypt1-3 cells expressing GFP-Atg8 are cultured in SMD selective medium at 25 °C to log phase. To mimic starvation, the cells are treated with rapamycin for 1hr at 37 °C.
2. Cells are centrifuged at 1500 x g for 2 min and approximately 2 μl of the cell pellet is examined on a slide.
3. Image acquisition of GFP-Atg8 is performed on an Applied Precision Delta Vison OMX Super Resolution System using a 100 x 1.4 NA oil-immersion objective and a Photometrics Evolve 512 EMCCD camera.
4. Images are taken and processed using Delta Vison OMX Master Control software and softWoRx reconstruction and analysis software. Autophagosome number is calculated from more than 300 cells in three separate experiments.
4. Notes
1. Prewash Ni-NTA agarose beads with 10 ml of bacterial lysis buffer 1 three times before use.
2. Prewash glutathione-sepharose beads with 10 ml of bacterial lysis buffer 2 three times before use.
3. Use blank beads to ensure each binding reaction has equal amounts of beads. To make blank beads, glutathione-sepharose beads are treated with 1 mg/ml BSA for 30 min and washed three times with 10 ml of PBS.
4. Other Rabs, such as Rab2A and Rab4A, are used as controls for in vitro binding experiments with Rab1A and CK1δ.
5. The beads are preincubated with 10 mg/ml of BSA for 1 hr before lysate is added.
6. Because Atg8 localizes to more than one structure, this analysis must be done in a ypt7Δ mutant background to ensure Atg8 labeled puncta are autophagosomes.
Acknowledgements
JW and SW received support from the National Natural Science Foundation of China (No. 91854115, No. 31970044 and No. 31771571). SF-N received support from the ALPHA-1 FOUNDATION and NIGMS under award numbers R01GM114111, R01GM115422 and R35GM131681.
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