Glycans function as a Golgi export signal to promote the constitutive exocytic trafficking

Most proteins in the secretory pathway are glycosylated. However, the role of glycans in membrane trafficking is still unclear. Here, we discovered that transmembrane secretory cargos, such as interleukin 2 receptor α subunit or Tac, transferrin receptor, and cluster of differentiation 8a, unexpectedly displayed substantial Golgi localization when their O-glycosylation was compromised. By quantitatively measuring their Golgi residence times, we found that the observed Golgi localization of O-glycan–deficient cargos is due to their slow Golgi export. Using a superresolution microscopy method that we previously developed, we revealed that O-glycan–deficient Tac chimeras localize at the interior of the trans-Golgi cisternae. O-Glycans were observed to be both necessary and sufficient for the efficient Golgi export of Tac chimeras. By sequentially introducing O-glycosylation sites to ST6GAL1, we demonstrated that O-glycan's effect on Golgi export is probably additive. Finally, the finding that N-glycosylated GFP substantially reduces the Golgi residence time of a Tac chimera suggests that N-glycans might have a similar effect. Therefore, both O- and N-glycans might function as a generic Golgi export signal at the trans-Golgi to promote the constitutive exocytic trafficking.


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Figure S1-8 Table S1 GFP- Tac  Tac-TC behaves like a Golgi resident. HeLa cells were used. (A) Example image showing the Golgi localization of GFP-Tac-STC. Cells transiently expressing GFP-Tac-STC were immunostained for endogenous GM130. (B,C) Tac-TC and GalT localize to the ER under brefeldin A treatment and to the recovered Golgi during the subsequent washout. Cells transiently coexpressing Sec61β-mCherry and GFP-Tac-TC (B) or GalT-GFP (C) were subjected to the indicated treatment and imaged live. The same images with different intensity scaling are shown in the second and third row to reveal the weak ER localization signal. (D) Tac-TC and GalT localize to the Golgi mini-stack under nocodazole treatment and to the recovered Golgi during the subsequent washout. Cells transiently co-expressing GFP-Tac-TC and GalT-mCherry were subjected to the indicated treatment and subsequently immuno-stained for endogenous GM130 (for images in the second row). In (B-D), boxed regions are enlarged at the upper right corner; scale bar, 20 μm.

Figure S2
Investigating the endocytic trafficking and Golgi residence times of secretory cargos and Golgi residents. HeLa cells were used. (A-C) Reporters employed in this study do not target to the Golgi from the PM or endolysosome. In (A), cells transiently expressing indicated fluorescence protein-tagged reporter were incubated with VHH-mCherry, rabbit anti-mCherry polyclonal antibody or mouse anti-CD8a monoclonal antibody for 2 h and subsequently subjected to immuno-staining of endogenous GM130 and the internalized antibody. CD8a-furin-mEOS2 was a positive control for the Golgi targeting from the PM or endolysosome. In (B,C), surfacelocalized ST-GFP and GFP-ERGIC53 are targeted to the endolysosome instead of the Golgi. Cells transiently expressing ST-GFP (B) or GFP-ERGIC53 (C) were incubated with VHH-mCherry for 2 h and subsequently subjected to immuno-staining of endogenous Lamp1 (a lysosome marker), CD63 (a late endosome marker) or EEA1 (an early endosome marker). Boxed regions were enlarged at the upper right corner. (D-M) Acquiring the Golgi residence times of Golgi residents and secretory cargos. These panels are related to Figure 2D. In (D-H), cells expressing indicated fluorescence protein-tagged reporter(s) were imaged live in the presence of CHX. Although not shown, GalT-mCherry was also co-expressed in (D,E). In (I-M), the corresponding total Golgi intensity was plotted as described in Figure 2C. Scale bar, 20 μm; error bar, mean ± standard error; n, the number of quantified cells.  The Golgi residence times and gel migration profiles of Tac mutants. (A) The schematic diagram showing the domain organization and glycosylation or mutation sites of C-terminally GFPtagged Tac and Tac(5A). The panel is organized as described in Figure 3A. (B,C) The Golgi residence time of Tac(5A)-GFP is significantly longer than that of Tac-GFP. The experiment and panel organization are described in Figure 3, B-E. Error bar, mean ± standard error; P values are from t test (unpaired and two-tailed); *****, P ≤ 0.000005; n, the number of quantified cells.       GFP(N-glyc)-Tac-TC is N-glycosylated. HeLa cells transiently expressing indicated construct were lysed and treated or not with PNGase F before gel separation and immuno-blotting for GFP-tag. Molecular weight (kDa) is indicated at the right side.  GFP-Tac(5A) localizes to the interior of the trans-Golgi cisternae. The experiment is similar to that of Figure 7D. Scale bar, 500 nm.     Uncropped gel images. Green box, the chemiluminescence image of the whole gel blot; black box, the cropped region for figure preparation; blue box, white light image of molecular weight marker bands, which are used to label the molecular weight (kDa). Fragment 1 consisting of the signal peptide was PCR amplified from Tac-GFP-intermediate using the primer pair #1. Fragment 2 consisting of the CDS of GFP was PCR amplified from pEGFP-N1 vector using the primer pair #2. Fragment 3 consisting of the CDS of Tac was PCR amplified from Tac-GFP-inermediate. Finanlly, the construct was assembled by the overlapping PCR of three fragments using the first and last listed primers and the PCR product was digested by NheI/EcoRI and ligated into pEGFP-C1 vector using the same sites.
GFP-Tac-STC GFP-Tac XhoI/EcoRI 5'-AGT GAC CTC GAG CTC GTC ACA ACA ACA GAT TTT C -3' and 5'-AGT GAC GAA TTC CTA GAT TGT TCT TCT ACT CTT CCT C -3' The CDS was PCR amplified from GFP-Tac using the listed primer pair. The PCR product was digested by XhoI/EcoRI and ligated into GFP-Tac using the same sites.
GFP-Tac-STC(5A) GFP-Tac XhoI/EcoRI 5'-AGT GAC CTC GAG CTC GTC ACA ACA GCA GAT TTT C -3' and 5'-AGT GAC GAA TTC CTA GAT TGT TCT TCT ACT CTT CCT C -3' The CDS was PCR amplified from GFP-Tac(5A) using the listed primer pair. The PCR product was digested by XhoI/EcoRI and ligated into GFP-Tac using the same sites. GFP- Tac Two PCRs were conducted using GFP-Tac as the template and Primer pair #1 and #2. The resulting two PCR fragments were mixed and subjected to the final PCR amplification using the first and last listed primers. The final PCR product was digested by XhoI/EcoRI and ligated into GFP-Tac using the same sites. Two PCRs were conducted using GFP-Tac as the template and Primer pair #1 and #2. The resulting two PCR fragments were mixed and subjected to the final PCR amplification using the first and last listed primers. The final PCR product was digested by AgeI/EcoRI and ligated into GFP-Tac using the same sites.
GFP-Cstem-Tac(5A) GFP-Tac XhoI/EcoRI Primer pair #1: 5'-AGT GAC CTC GAG CCA GCG AAG CCC ACC ACG ACG -3', 5'-CGG GTC ATC GTC ACA GAG CTC CAG CCC CCT CGT GTG CAC -3' and Primer pair #2: 5'-GTG CAC ACG AGG GGG CTG GAG CTC TGT GAC GAT GAC CCG -3', 5'-AGT GAC GAA TTC CTA GAT TGT TCT TCT ACT CTT CCT C -3' Two PCRs were conducted using CD8a and GFP-Tac(5A) as the template and Primer pair #1 and #2 respectively. The resulting two PCR fragments were mixed and subjected to the final PCR amplification using the first and last listed primers. The final PCR product was digested by XhoI/EcoRI and ligated into GFP-Tac using the same sites. Two PCRs were conducted using CD8a as the template and Primer pair #1 and #2. The resulting two PCR fragments were mixed and subjected to the final PCR amplification using the first and last listed primers. The final PCR product was digested by XhoI/EcoRI and ligated into GFP-Tac using the same sites.