Fig. 1 Tunicamycin strongly inhibits glycoprotein secretion Fig. 1 Tunicamycin strongly inhibits glycoprotein secretion. Tobacco cells were pre-incubated with 18 µM of tunicamycin (Tm) for 90 min. The cells were pulse-labeled for 6 h with either [<sup>35</sup>S]methionine and [<sup>35</sup>S]cysteine (lanes 1–4) or [<sup>3</sup>H]glucosamine (lanes 5–8). At the end of the treatment, proteins from intracellular (MI) and extracellular (MEX) protein extracts were analysed by SDS-PAGE and fluorography. From: Fusion with HDEL Protects Cell Wall Invertase from Early Degradation when N-glycosylation is Inhibited Plant Cell Physiol. 2003;44(2):173-182. doi:10.1093/pcp/pcg027 Plant Cell Physiol |
Fig. 2 Effects of tunicamycin on the secretion of endogenous tobacco and recombinant carrot cell wall invertases. Transgenic cells were pre-incubated with either no inhibitor or with 18 µM of tunicamycin for 90 min, pulsed with [<sup>35</sup>S]methionine and [<sup>35</sup>S]cysteine for 90 min and chased for 0, 2, 4, 8 h in presence (Tunicamycin) or in the absence (Control) of the drug. Endogenous tobacco and recombinant carrot invertases were immunoprecipitated from microsomal (MI) and extracellular (MEX) protein extracts, analysed on SDS-PAGE and fluorography. Fluorograph obtained after treatment with tunicamycin was exposed four times longer than control to facilitate invertases quantification. Arrowheads indicate the position of a 68 kDa protein marker and asterisks indicate the position of non-glycosylated tobacco and recombinant carrot cell wall invertases. From: Fusion with HDEL Protects Cell Wall Invertase from Early Degradation when N-glycosylation is Inhibited Plant Cell Physiol. 2003;44(2):173-182. doi:10.1093/pcp/pcg027 Plant Cell Physiol |
Fig. 3 Transport of PHA and sporamin from the microsomes to the vacuole in tunicamycin-treated tobacco cells. Transgenic tobacco cells were pretreated with 18 µM of tunicamycin (TM) for 90 min, pulsed with [<sup>35</sup>S]methionine and [<sup>35</sup>S]cysteine for 90 min and chased for times indicated on the right of each panel. Microsomal (MI) and soluble (S) proteins were separated by size exclusion chromatography on a Sepharose-4B column. PHA and sporamin (SPO) were immunoprecipitated from each fraction and analysed on SDS-PAGE and fluorography. From: Fusion with HDEL Protects Cell Wall Invertase from Early Degradation when N-glycosylation is Inhibited Plant Cell Physiol. 2003;44(2):173-182. doi:10.1093/pcp/pcg027 Plant Cell Physiol |
Fig. 4 Effects of wortmannin on the vacuolar transport of protein markers in tunicamycin-treated tobacco cells. Cells were pretreated with either no inhibitor, 18 µM of tunicamycin (TM) for 90 min, 33 µM of wortmannin (WORT) for 30 min or with both tunicamycin and wortmannin (lanes 4 and 8). Cells were pulsed with [<sup>35</sup>S]methionine and [<sup>35</sup>S]cysteine for 90 min and chased for 18 h in presence of the drugs. PHA, sporamin (SPO), chitinase (CHIT) and β-1,3-glucanase (GLUC) were immunoprecipitated from the intracellular (MI) and extracellular (MEX) protein extracts and analysed by SDS-PAGE and fluorography. From: Fusion with HDEL Protects Cell Wall Invertase from Early Degradation when N-glycosylation is Inhibited Plant Cell Physiol. 2003;44(2):173-182. doi:10.1093/pcp/pcg027 Plant Cell Physiol |
Fig. 5 Tunicamycin does not prevent Δprosporamin secretion Fig. 5 Tunicamycin does not prevent Δprosporamin secretion. Transgenic tobacco cells were pre-incubated either with no inhibitor or with 18 µM of tunicamycin for 90 min, pulsed with [<sup>35</sup>S]methionine and [<sup>35</sup>S]cysteine and chased for 0, 4, 8, 12, 24 h in presence (Tunicamycin) or in the absence (Control) of the drug. Δprosporamin was immunoprecipitated from intracellular (MI) and extracellular (MEX) protein extracts. The polypeptides were analysed on SDS-PAGE and fluorography. Arrowheads indicate the position of a 29 kDa molecular mass marker. From: Fusion with HDEL Protects Cell Wall Invertase from Early Degradation when N-glycosylation is Inhibited Plant Cell Physiol. 2003;44(2):173-182. doi:10.1093/pcp/pcg027 Plant Cell Physiol |
Fig. 6 Analysis of non-glycosylated cell wall invertases degradation in tobacco cells treated simultaneously with tunicamycin and monensin. Tobacco cells were pre-incubated with either no inhibitor (lanes 1 and 2) or 18 µM tunicamycin (TM) for 90 min (lanes 3 and 4), 5 µM of monensin (MON) for 30 min (lanes 5 and 6) or both tunicamycin (18 µM) and monensin (5 µM) (lanes 7 and 8). Cells were pulsed with [<sup>35</sup>S]methionine and [<sup>35</sup>S]cysteine for 90 min and chased for 0 or 4 h in presence of the drugs. Endogenous tobacco and recombinant carrot cell wall invertases were immunoprecipitated from the microsomal (MI) and extracellular (MEX) protein extracts, analysed by SDS-PAGE and fluorography. From: Fusion with HDEL Protects Cell Wall Invertase from Early Degradation when N-glycosylation is Inhibited Plant Cell Physiol. 2003;44(2):173-182. doi:10.1093/pcp/pcg027 Plant Cell Physiol |
Fig. 7 Analysis of non-glycosylated cell wall invertases degradation in tobacco cells treated simultaneously with tunicamycin and BFA. Tobacco cells were pre-incubated with either no inhibitor (lanes 1 and 2) or 18 µM tunicamycin (TM) for 90 min (lanes 3 and 4), 36 µM of BFA for 60 min (lanes 5 and 6) or both tunicamycin (18 µM) and BFA (36 µM) (lanes 7 and 8). Cells were pulsed with [<sup>35</sup>S]methionine and [<sup>35</sup>S]cysteine for 90 min and chased for 0 or 4 h in presence of the drugs. Endogenous tobacco and recombinant carrot cell wall invertases were immunoprecipitated from the microsomal (MI) and extracellular (MEX) protein extracts, analysed by SDS-PAGE and fluorography. From: Fusion with HDEL Protects Cell Wall Invertase from Early Degradation when N-glycosylation is Inhibited Plant Cell Physiol. 2003;44(2):173-182. doi:10.1093/pcp/pcg027 Plant Cell Physiol |
Fig. 8 The effects of proteasome inhibitors on non-glycosylated cell wall invertases degradation in presence of tunicamycin. Tobacco cells were pre-incubated with either no inhibitor (lanes 1 and 2) or 18 µM of tunicamycin (TM) for 90 min (lanes 3 and 4), 50 µM of CBZ for 60 min (lanes 5 and 6), 250 µM of LLnL for 60 min (lanes 7 and 8), both tunicamycin (18 µM) and CBZ (50 µM) (lanes 9 and 10), or both tunicamycin (18 µM) and LLnL (250 µM) (lanes 11 and 12). Cells were with [<sup>35</sup>S]methionine and [<sup>35</sup>S]cysteine for 90 min and chased for 0 or 4 h in presence of the drugs. Endogenous tobacco and recombinant carrot cell wall invertases were immunoprecipitated from the microsomal (MI) and extracellular (MEX) protein extracts, analysed by SDS-PAGE and fluorography. From: Fusion with HDEL Protects Cell Wall Invertase from Early Degradation when N-glycosylation is Inhibited Plant Cell Physiol. 2003;44(2):173-182. doi:10.1093/pcp/pcg027 Plant Cell Physiol |
Fig. 9 Retention in the ER stabilizes non-glycosylated cell wall invertase. Tobacco cells expressing carrot cell wall invertase fused with an HDEL, ER retention signal were pre-incubated with either no inhibitor or with [<sup>35</sup>S]methionine and [<sup>35</sup>S]cysteine for 90 min and chased for 0, 3, 6, 12, 24 h in presence (+) or in absence (–) of the drug. Recombinant carrot invertase was immunoprecipitated from the microsomal fraction and analysed on SDS-PAGE and fluorography. Arrowhead indicates the position of 68 kDa protein marker. From: Fusion with HDEL Protects Cell Wall Invertase from Early Degradation when N-glycosylation is Inhibited Plant Cell Physiol. 2003;44(2):173-182. doi:10.1093/pcp/pcg027 Plant Cell Physiol |