Regulation of protein glycosylation and sorting by the Golgi matrix proteins GRASP55/65

Abstract

The Golgi receives the entire output of newly synthesized cargo from the endoplasmic reticulum, processes it in the stack largely through modification of bound oligosaccharides, and sorts it in the trans-Golgi network. GRASP65 and GRASP55, two proteins localized to the Golgi stack and early secretory pathway, mediate processes including Golgi stacking, Golgi ribbon linking and unconventional secretion. Previously, we have shown that GRASP depletion in cells disrupts Golgi stack formation. Here we report that knockdown of the GRASP proteins, alone or combined, accelerates protein trafficking through the Golgi membranes but also has striking negative effects on protein glycosylation and sorting. These effects are not caused by Golgi ribbon unlinking, unconventional secretion or endoplasmic reticulum stress. We propose that GRASP55/65 are negative regulators of exocytic transport and that this slowdown helps to ensure more complete protein glycosylation in the Golgi stack and proper sorting at the trans-Golgi network.

Figure 1. Depletion of GRASP55/65 does not cause apoptosis or ER stress

(A) HeLa cells transfected with the indicated RNAi were stained with annexin V-GFP before fixation. Note that no obvious annexin V staining was observed on GRASP-depleted cells. (B) HeLa cells treated with 2 µM staurosporine (Stau) for 0 h or 2 h were stained with annexin V-GFP before fixation. Scale bars in A-B, 10 µm. (C) HeLa cells transfected with the indicated RNAi or treated with staurosporine (ST) were analyzed by Western blot. Note that the cleaved caspase 3 was detected after 2 h of staurosporine treatment, but not following the depletion of the GRASPs. (D) Depletion of GRASPs does not cause ER stress. HeLa cells transfected with the indicated RNAi or treated with DMSO or 2 µM thapsigargin (Thapsig) for 4 h or 16 h were analyzed by SDS-PAGE and Western blot. Note that the protein level of Bip increased after thapsigargin treatment, but not following GRASP depletion. (E) GRASP depletion does not result in ERAD. HeLa cells transfected with indicated RNAi were first treated with 5 µg/ml BFA for 1 h followed by the addition of 100 µM cycloheximide (CHX) into the medium and incubated for the indicated time period. Shown are Western blots of cell lysate for β1 integrin and p97 on the same gels. Molecular weight standards (kDa) are indicated on the left in C–E.

Figure 2. Depletion of GRASPs accelerates α5 integrin trafficking

(A) GRASP depletion increases α5 integrin trafficking. HeLa cells transfected with the indicated RNAi were labeled with 250 µCi/ml TRANS ³⁵S-LABEL for 1 h, chased for the indicated time periods, and lysed. α5 integrin were immunoprecipitated and analyzed by SDS-PAGE and autoradiography. Note that the mature form of α5 integrin appeared more rapidly in GRASP-depleted cells. (B) The ratio of the mature form vs. total α5 integrin at the indicated chase time for one representative result out of three independent experiments. Shown are the quantitation results from three independent experiments. P-value was determined by Student’s t-test; *, p < 0.05; **, p<0.01; ***, p<0.001 in this and all following figures. Note that the maturation rate of α5 integrin increased in GRASP-depleted cells.

Figure 3. GRASP depletion accelerates VSV-G trafficking through the Golgi membranes

(A) VSV-G traffic to the cell surface is accelerated by GRASP depletion. HeLa cells stably expressing VSV-G-ts045-GFP under an inducible promoter were transfected with control (ctrl), GRASP55 (55), GRASP65 (65), or both GRASP55 and GRASP65 (55+65) RNAi. The cells were incubated with doxycycline at 40.5°C for 16 h to induce VSV-G expression and shifted to 32°C for 120 min to release the VSV-G from the ER. The cells were collected, and the VSV-G on the surface was stained with an antibody against its extracellular domain followed by incubation with fluorescently labeled secondary antibodies. Total VSV-G and surface-bound VSV-G were assessed by flow cytometry. The ratio of surface-to-total VSV-G normalized against control cells (mean±SEM) are shown based on three independent experiments. (B) GRASP depletion accelerates VSV-G trafficking. HeLa cells transfected with indicated RNAi were infected with VSV-G ts045-GFP adenovirus and incubated at 40.5°C for 16 h. Cells were shifted to 32°C, incubated for indicated time period (chase), and lysed. Proteins were treated with EndoH and analyzed by Western blot for VSV-G-GFP. Arrows indicate the EndoH resistant form and arrowheads indicate the EndoH sensitive form of VSV-G. Molecular weight markers are indicated on the left. (C) Quantitation of (B) to indicate the percentage of VSV-G in EndoH resistant form. Note that the difference between the GRASP-depleted cells and control RNAi treated cells is most dramatic after 15–45 min chase when VSV-G travels through the Golgi membranes. (D) GRASP depletion accelerates VSV-G trafficking through the Golgi membranes. HeLa cells transfected with indicated RNAi were infected with VSV-G ts045-GFP adenovirus and incubated at 40.5°C for 16 h. Cells were shifted to 15°C for 2 h and then to 20°C for 0 and 10 min followed by immunofluorescence microscopy. The colocalization of VSV-G with GalT was quantified with ImageJ. Shown are representative results (mean±SEM from 20 different cells) from three independent experiments.

Figure 4. GRASP depletion enhances membrane association of coat proteins

HeLa cells transfected with indicated RNAi were subjected to subcellular fractionation. Proteins in the cytosol and membrane fractions were analyzed by Western blot for β-COP (A), δ-COP (B), clathrin heavy chain (CHC, C), sec31 (D), bet1 (E) and α-tubulin (F). In some panels 3 times of the membrane fractions were loaded onto the SDS-PAGE (lane 3). Shown are representative images from 3 independent experiments. Quantification results (mean±SEM) are shown on the right to indicate the percentage of the protein found in the membrane fractions. (G) GRASP depletion causes vesiculation of the Golgi. Quantitation of EM images of GRASP-depleted cells for the number of vesicles adjacent to a Golgi stack. Shown are representative results (mean±SEM from 20 different cells) from three independent experiments. Cells with both GRASP55 and GRASP65 depleted did not usually contain distinguishable Golgi stacks and thus were not quantified.

Figure 5. Glycomic analysis of total protein glycosylation in GRASP-depleted cells

HeLa cells expressing RNAi targeting the indicated GRASP proteins were collected and processed for glycomic analysis by mass spectrometry. (A) The major high-mannose and complex glycans were quantified relative to an external standard and normalized to protein content. Double knockdown cells exhibit significantly decreased glycan abundance. Glycan structural representations are consistent with the nomenclature proposed by the Consortium for Functional Glycomics: GlcNAc, blue square; Man, green circle; Gal, yellow circle; SA (sialic acid), purple diamond; Fuc, red triangle. (B) The fold-change for each glycan indicated in panel A was calculated (knockdown/control). Average fold-changes ± SEM are plotted; negative values indicate decreased abundance relative to control. Single knockdowns exhibit relatively minor alteration in the pool of major high-mannose and complex glycans, but the double knockdown cells are significantly reduced in both high-mannose and complex glycans. In the double knockdown cells, the fold-reduction in complex glycans is greater than the reduction in high-mannose glycans (Student’s t-test, **, p<0.001). (C) Lipid-linked oligosaccharides (LLO) and free oligosaccharides (FOS) were quantified by mass spectrometry relative to an external standard and normalized to protein content (Dol, dolichol pyrophosphate). Total LLO levels are significantly lower than FOS levels, note change in y-axis scale. All quantifiable LLO species were reduced in the GRASP double knockdown cells, while some LLOs increased and some decreased relative to control in the single knockdown cells. The abundance of all quantifiable FOS species was dramatically increased in double knockdown cells, while single knockdown had relatively little effect on FOS levels. (D) Fold-changes in LLO and FOS, calculated as in panel B, reveals the inverse relationship expected for precursor and product. Comparison to the fold-change in glycoprotein glycan abundance (panel B) emphasizes that LLO availability limits glycosylation. When LLOs are decreased by knockdown, glycoprotein glycosylation is also decreased (55 alone and 55+65) and vice versa (65 alone), although the impact of either single knockdown is much smaller than the double knockdown.

Figure 6. Depletion of GRASP results in altered cell-surface glycosylation

(A–D) Surface staining of fluorophore-conjugated lectin MAA specific for α(2,3) sialic acid was reduced upon the depletion of GRASPs. Non-permeabilized HeLa cells treated with the indicated RNAi were exposed to TRITC-conjugated MAA. The cells were then fixed, stained with GRASP antibodies, and visualized using a confocal microscope. Scale bar, 50 µm. (E–F) HeLa cells transfected with control RNAi (arrows) and with RNAi for GRASP65 and 55 (asterisks) were mixed, cultured together and stained for GRASP65 (green) and WGA (red). Scale bar, 20 µm. (G) GRASP depletion impacts Lamp1 and Lamp2 glycosylation. Indicated GRASP-depleted cells were analyzed for Lamp1, Lamp2 and p97 by Western blots. Molecular weight markers are shown on the right. Note the shift of the bands after GRASP depletion.

Figure 7. GRASP depletion does not affect the localization of Golgi enzymes

HeLa cells were treated with the indicated RNAi and analyzed by immunofluorescence microscopy for Mannosidase II (ManII, A), Galactosyltransferase (GalT, B) and GM130. Note that Golgi enzymes colocalized with GM130 in all cells. Scale bar, 10 µm.

Figure 8. GRASP depletion impairs cathepsin D sorting

(A) Missorting of cathepsin D in cells by GRASP depletion. HeLa cells transfected with the indicated RNAi were washed with PBS and incubated in serum-free DMEM for 2 h. The secreted proteins in the medium were TCA precipitated and normalized based on the total protein in the cell lysate. Equal portions of the cell lysate and medium were analyzed by Western blot for cathepsin D. Immature form: 53 kDa; intermediate form: 47 kDa; mature form: 31 kDa. The asterisk indicates a nonspecific band that appears in the medium. (B) Ratio of intermediate vs. immature cathepsin D in cells transfected with the indicated RNAi by quantifying the related bands on the Western blots in (A). (C) The amount of cathepsin D in the cell lysate normalized to the cells transfected with control RNAi. (D) The amount of immature cathepsin D in the medium normalized to the cells transfected with control RNAi. The data are presented as the mean±SEM of four independent experiments.