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. 2006 Dec;17(12):5153-62.
doi: 10.1091/mbc.e06-08-0665. Epub 2006 Oct 4.

Transition of galactosyltransferase 1 from trans-Golgi cisterna to the trans-Golgi network is signal mediated

Beat E Schaub  1 , Bea Berger, Eric G Berger, Jack Rohrer
Affiliations

Transition of galactosyltransferase 1 from trans-Golgi cisterna to the trans-Golgi network is signal mediated

Beat E Schaub et al. Mol Biol Cell. 2006 Dec.

Abstract

The Golgi apparatus (GA) is the organelle where complex glycan formation takes place. In addition, it is a major sorting site for proteins destined for various subcellular compartments or for secretion. Here we investigate beta1,4-galactosyltransferase 1 (galT) and alpha2,6-sialyltransferase 1 (siaT), two trans-Golgi glycosyltransferases, with respect to their different pathways in monensin-treated cells. Upon addition of monensin galT dissociates from siaT and the GA and accumulates in swollen vesicles derived from the trans-Golgi network (TGN), as shown by colocalization with TGN46, a specific TGN marker. We analyzed various chimeric constructs of galT and siaT by confocal fluorescence microscopy and time-lapse videomicroscopy as well as Optiprep density gradient fractionation. We show that the first 13 amino acids of the cytoplasmic tail of galT are necessary for its localization to swollen vesicles induced by monensin. We also show that the monensin sensitivity resulting from the cytoplasmic tail can be conferred to siaT, which leads to the rapid accumulation of the galT-siaT chimera in swollen vesicles upon monensin treatment. On the basis of these data, we suggest that cycling between the trans-Golgi cisterna and the trans-Golgi network of galT is signal mediated.

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Figures

Figure 1.
Figure 1.
Double-staining of galT-GFP and siaT-GFP with endogenous galT and siaT in HepG2 cells treated with monensin. GalT-GFP and siaT-GFP colocalize with their endogenous counterpart in HepG2 cells. (A) In the GA of control cells, stably transfected galT-GFP (green) colocalizes with endogenous galT (red) labeled with mAb against galT in the GA. (B) On treatment with 2 μM monensin for 30 min both galT-GFP (green) and endogenous galT (red) are redistributed to swollen vesicles (arrowheads). (C) In control cells, stably transfected siaT-GFP (green) colocalizes with endogenous siaT (red) labeled with polyclonal antibody against siaT. (D) On treatment with 2 μM monensin for 30 min both siaT-GFP (green) and endogenous siaT (red) remain together and show little change. Bar, 10 μm.
Figure 2.
Figure 2.
Double-staining time-lapse videomicroscopy of galT-GFP and siaT-RFP in HepG2 cells treated with monensin. GalT-GFP rapidly separates from siaT-RFP and endogenous giantin upon monensin treatment. (A) HepG2 cells stably transfected with galT-GFP (green) were transiently transfected with siaT-RFP (red). Seventy-two hours after transfection of siaT-RFP, monensin was added to a final concentration of 2 μM, and the movie (3 frames/min) was recorded on a confocal microscope, starting ∼1 min after addition of monensin. Bar, 10 μm. See also Supplementary Movie S1. (B and C) Double immunofluorescence of galT-GFP (green) and giantin (red) in control cells (B) and cells treated with 2 μM monensin for 30 min (C). (D and E) Double immunofluorescence of siaT-GFP (green) and giantin (red) in control cells (D) and cells treated with 2 μM monensin for 30 min (E). Bar, 10 μm.
Figure 3.
Figure 3.
Dynamics of galT-GFP and siaT-GFP in monensin-treated cells followed by addition of BFA in HepG2 cells. The time-lapse video starts immediately after addition of 10 μM BFA (0 min). (A and C) Control experiments in absence of monensin; both galT-GFP (A, Supplementary Movie S2) and siaT-GFP (C, Supplementary Movie S4) relocated to the ER after treatment with 10 μM BFA. (B and D) Fifteen-minute monensin (2 μM) pretreatment: galT-GFP (B, Supplementary Movie S3) is present in swollen vesicles (arrowheads) at the time of addition of BFA and remains in swollen vesicles. (D) Supplementary Movie S5: SiaT-GFP is relocated to the ER also in presence of 2 μM monensin. Bar, 10 μm.
Figure 4.
Figure 4.
Double-staining of galT-GFP or siaT-GFP with TGN6 in monensin-treated HepG2 cells. GalT-GFP but not siaT-GFP can be found in TGN46-positive swollen vesicles. HepG2 cells stably transfected with galT-GFP or siaT-GFP were immunolabeled using anti TGN46 antibody. (A and C) Significant overlap between TGN46 (red) and galT-GFP (A, green) or siaT-GFP (C, green) in the Golgi region, with additional peripheral staining of TGN46, possibly in endosomes. (B) On treatment with 2 μM monensin for 30 min both galT-GFP and TGN46 are found in swollen vesicles (arrowheads). (D) Although treatment of the cells with 2 μM monensin for 30 min does not affect siaT-GFP localization, TGN46 is redistributed to swollen vesicles, and the Golgi region stained with siaT-GFP is devoid of TGN46. Bar, 10 μm.
Figure 5.
Figure 5.
Optiprep density gradient of HepG2 cells treated with monensin. Fraction 1 contains densest membranes; fraction 10 contains lightest membranes. GalT-GFP (A) as well as siaT-GFP (C) from control are enriched in fractions 5 and 6. (B) GalT-GFP from cells treated with 2 μM monensin for 30 min are distributed in fractions 4–10. (D) SiaT-GFP from cells treated with 2 μM monensin for 30 min remains unchanged and remain enriched in fractions 5 and 6. For densitometric analysis, see Table 1.
Figure 6.
Figure 6.
Chimeras of galT and siaT fused to GFP. For easy reference the constructs were designated with a three-letter code for the three domains possibly involved (cytoplasmic tail, transmembrane domain, and stem region), indicating the origin of the domain: G for galT derived and S for siaT derived. The numbers refer to the amino acid number of the respective full-length protein (galT, GenBank accession no. NP_001497.2; siaT, GenBank accession no. BC040009).
Figure 7.
Figure 7.
Response of various galT/siaT–GFP chimeras to monensin. (A and C) control; (B and D) monensin-treated HepG2 cells (2 μM monensin, 30 min). Double-immunofluorescent pictures of SGG-GFP (A and B, green) and GSS-GFP (C and D, green), and giantin (red). (A and C) The GFP-chimeras and giantin colocalize under control conditions. (B) SGG-GFP remains colocalized with giantin in monensin-treated cells. (D) In monensin-treated cells, GSS-GFP separates from giantin and is found in swollen vesicles. (E–H) Optiprep density gradients. SGG-GFP (E and F) and GSS-GFP (G and H). (E and G) Both SGG-GFP (E) and GSS-GFP (G) are enriched in fractions 5 and 6 in untreated cells. (F) SGG-GFP remains enriched in fractions 5 and 6 after treatment with 2 μM monensin for 30 min. (H) GSS-GFP shifts toward less dense fractions upon treatment of the cells with 2 μM monensin for 30 min.
Figure 8.
Figure 8.
Scheme of mutants of galT and response to monensin of the long iso-form. (A) Schematic drawing of the various chimeras. (B) Stably transfected galTshort-GFP (green) colocalizes with immunostained endogenous galT (red) in control cells. (C) On monensin treatment endogenous galT is found in swollen vesicles, whereas galTshort-GFP is mostly found in the Golgi.
Figure 9.
Figure 9.
Fusion of the cytoplasmic tail of galT to siaT-GFP confers monensin sensitivity. (A and C) In the GA of control cells, stably transfected GSSS-GFP (A, green) and GSGG-GFP (C, green) colocalize with endogenous giantin (red). (B and D) On treatment with 2 μM monensin for 30 min both GSSS-GFP (B, green) and GSGG-GFP (D, green) dissociate from giantin (red) and are redistributed to swollen vesicles. Bar, 10 μm.
Figure 10.
Figure 10.
Model for GA-to-TGN transition. In the steady state, long and short iso-forms of galT as well as siaT are accumulated in the trans-Golgi. GalT is assumed to constantly cycle from the trans-Golgi to the TGN and back to nondefined proximal sites of the secretory pathway. In presence of monensin, galT is arrested in TGN-derived swollen vesicles where it colocalizes with TGN46. Anterograde transport from those sites is retarded only (Strous et al., 1985), whereas retrograde transport is blocked, permitting accumulation in swollen vesicles.
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