GRASP55 restricts early-stage autophagy and regulates spatial organization of the early secretory network

Abstract

There is great interest in understanding the cellular mechanisms controlling autophagy, a tightly regulated catabolic and stress-response pathway. Prior work has uncovered links between autophagy and the Golgi reassembly stacking protein of 55 kDa (GRASP55), but their precise interrelationship remains unclear. Intriguingly, both autophagy and GRASP55 have been functionally and spatially linked to the endoplasmic reticulum (ER)---Golgi interface, broaching this compartment as a site where GRASP55 and autophagy may intersect. Here, we uncover that loss of GRASP55 enhances LC3 puncta formation, indicating that GRASP55 restricts autophagosome formation. Additionally, using proximity-dependent biotinylation, we identify a GRASP55 proximal interactome highly associated with the ER-Golgi interface. Both nutrient starvation and loss of GRASP55 are associated with coalescence of early secretory pathway markers. In light of these findings, we propose that GRASP55 regulates spatial organization of the ER-Golgi interface, which suppresses early autophagosome formation.

Keywords: Autophagy; Cell biology; GRASP55.

Fig. 1.

Effect of GRASP55 depletion on autophagy. (A) GRASP55 knockdown (shG55 #06 or #63) or non-targeting control (shNT) cells were incubated in EBSS for 60 min with 100 nM bafilomycin A1 (BafA1) or left untreated (UT). Cells were immunostained for LC3B and DAPI-counterstained. Representative images are shown. Scale bar: 10 μm. (B) LC3B puncta were enumerated and normalized to the number of DAPI-stained nuclei on a per-image basis. Welch's ANOVA with Games-Howell's multiple comparisons test was performed within each treatment group, and multiplicity adjusted P-values are shown. n=90 images per group pooled from three replicates. Error bars: mean±95% confidence interval. (C) GRASP55 knockout (G55 KO) and control cells stably expressing mCherry-EGFP-LC3B were incubated in glucose-free media for 4 h. Live cells were imaged. Representative images are shown. Scale bar: 10 μm. (D) Double-positive (mCherry⁺EGFP⁺), single-positive (mCherry⁺EGFP⁻), and total (mCherry⁺EGFP⁺+mCherry⁺EGFP⁻) LC3B puncta were counted and normalized to the number of nuclei on a per-image basis. A two-tailed unpaired Welch's t-test was performed for each group, and P-values are shown. n=77 images per cell line pooled from three replicates. Error bars: mean±95% confidence interval. (E) The ratio of mCherry⁺EGFP⁺ puncta to total puncta on a per-image basis was calculated and reported as a percentage. A two-tailed unpaired Welch's t-test was performed, and the P-value is shown. n=77 images per cell line pooled from three replicates. Error bars: mean±95% confidence interval. (F) GRASP55 knockdown and control cells were incubated in EBSS for 60 min with 100 nM BafA1 or left untreated. Lysates were analyzed by anti-LC3 immunoblotting. A representative immunoblot is shown. (G) Densitometry of immunoblots from Fig. 1F. LC3-II/GAPDH ratios were calculated, normalizing to shNT controls within each treatment group. One-sample two-tailed t-tests were performed with a test value of 1, and P-values are shown. n=4 bioreplicates. Error bars: mean±s.d.

Fig. 2.

GRASP55 proximity-dependent biotinylation. (A) HEK293T cells stably expressing GRASP55^WT-BirA*-HA or GRASP55^G2A-BirA*-HA were pulsed with 50 μM biotin to induce proximity-dependent biotinylation. Biotinylated proteins were affinity purified from lysates using NeutrAvidin beads and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by by label-free quantification (LFQ) using MaxQuant software. (B) Hits from the GRASP55 proximal interactome were analyzed for functional protein-protein interactions (PPI) using the STRING database tool. Putative functional relationships are indicated by network edges connecting protein nodes, with strength of support represented by line thickness. The PPI enrichment P-value reported by STRING is shown. (C,D) Gene ontology enrichment analysis of cellular compartment and biological process terms. A Fisher's exact test with false discovery rate (FDR) correction was performed to evaluate statistical significance. Fold enrichment of the most specific subclass terms using the hierarchy sort function with FDR <0.05 are shown, along with individual FDR values. *: greater than 100-fold enrichment. ER: endoplasmic reticulum; ERGIC: endoplasmic reticulum-Golgi intermediate compartment. (E,F,G) Immunoblots for selected proteins from the GRASP55 proximal interactome were performed. HEK293T cells stably expressing wild-type (G55^WT-BirA*-HA, WT) or mutant (G55^G2A-BirA*-HA, G2A) GRASP55 BioID constructs or empty vector control (EV) were incubated with 50 μM biotin for 24 h in full growth media (FGM) or serum-free media (SFM). Whole cell lysates (WCL) were harvested and affinity purified (AP) for biotinylated proteins, and immunoblot analysis was performed as indicated. Representative immunoblots are shown. Band densitometry was performed on AP samples, and the WT to G2A band intensity ratios were log₂-transformed. One-sample two-tailed t-tests were performed with a test value of 0, and P-values are shown. n=3 bioreplicates. Error bars: mean±s.d. H. GRASP55 knockdown HEK293T cells (shG55 #06) were transiently depleted of selected proteins from the GRASP55 proximal interactome using siRNAs against Sec23IP, Sec24B, and TRIP11 or a non-targeting (NT) control. Double-knockdown cells were incubated in EBSS with 100 nM bafilomycin A1 (BafA1) for 60 min and stained for LC3B along with DAPI. Representative images are shown. Scale bar: 10 μm. I. LC3B puncta were counted and normalized to the number of DAPI-stained nuclei on a per-image basis. Welch's ANOVA with Games-Howell's multiple comparisons test was performed, and multiplicity adjusted P-values are shown. n=150 images per group pooled from three replicates. Error bars: mean±95% confidence interval.

Fig. 3.

Effect of starvation on the ER-Golgi interface. (A) HEK293T cells were incubated in EBSS for 1.5 h or maintained in full growth media (FGM) and stained for GRASP55, GM130, and Sec16A along with DAPI. Representative images are shown. Scale bar: 10 μm. (B) HEK293T cells were incubated in EBSS for indicated times or maintained in FGM and stained for GM130 and Sec24B along with DAPI. Representative images are shown. Scale bar: 10 μm. (C) Analysis of immunofluorescence results from Fig. 3A. Areas covered by GRASP55 (G55), GM130, and Sec16A as well as the overlapping regions between GRASP55 or GM130 with Sec16A were measured using ImageJ. The ratio of overlapping region to individual area of a structure was calculated as indicated and reported as a percent. Unpaired two-tailed t-tests were performed, and P-values are shown. n=74 FGM cells and 76 EBSS cells, pooled from two experiments. Error bars: mean±95% confidence interval. (D) Analysis of immunofluorescence results from Fig. 3B. The areas covered by GM130 and Sec24B, including overlapping regions, were measured using ImageJ. The ratio of overlapping region to individual area of a structure was calculated and reported as a percent. One-way ANOVA with Dunnett's multiple comparisons test was performed for each colocalization group, and multiplicity adjusted P-values are shown. n=149 FGM cells, 150 EBSS 2 h cells, and 150 EBSS 6 h cells, pooled from three replicates. Error bars: mean±95% confidence interval.

Fig. 4.

Effect of GRASP55 depletion on the ER-Golgi interface. (A) GRASP55 stable knockdown (shG55 #06 and #63) and non-targeting control (shNT) cells were stained for GM130 and Sec24B along with DAPI. Representative images are shown. Scale bar: 10 μm. (B) The areas covered by GM130 and Sec24B, including overlapping regions, were measured using ImageJ software. The ratio of overlapping region to individual area of a structure was calculated and reported as a percent. Welch's ANOVA with Games-Howell's multiple comparisons test was performed for each colocalization group, and multiplicity adjusted P-values are shown. n=150 cells pooled from three replicates. Error bars: mean±95% confidence interval.