Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor.

Kim, Jae Hong; Lingwood, Clifford A.; Williams, David B.; Furuya, Wendy; Manolson, Morris F.; Grinstein, Sergio

doi:10.1083/jcb.134.6.1387

Cited by 137 publications

(138 citation statements)

References 38 publications

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“…However, no obvious colocalization between B-Glyc-KDEL and the lysosomal marker Lamp-2 was detected. These data are consistent with a recent report on intracellular transport of verotoxin-1 B-fragment (100% similar to Shiga toxin B-fragment) in which no accumulation of B-fragment in lysosomal structures has been detected (40). Interestingly, we found that BFA, which was shown to have only limited effects on transport to lysosomes (49 -52), not only completely abolished glycosylation of B-Glyc-KDEL, but also strongly inhibited the appearance of the putative degradation product of B-Glyc-KDEL.…”

Section: Discussionsupporting

confidence: 82%

“…4, G and H). The same result was obtained for DTAF-labeled wild type B-fragment (not shown), which is in good agreement with previously published experiments on B-fragment transport to the Golgi apparatus (40). However, a fraction of B-Glyc-KDELGL was present in the ER, as documented by colocalization with signal sequence receptor protein (Fig.…”

Section: Fig 4 Colocalization Of B-glyc-kdel and B-glyc-kdelgl Withsupporting

confidence: 81%

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Retrograde Transport of KDEL-bearing B-fragment of Shiga Toxin

Johannes¹,

Tenza

Antony

et al. 1997

Journal of Biological Chemistry

187

254

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To investigate retrograde transport along the biosynthetic/secretory pathway, we have constructed a recombinant Shiga toxin B-fragment carrying an N-glycosylation site and a KDEL retrieval motif at its carboxyl terminus (B-Glyc-KDEL). After incubation with HeLa cells, B-Glyc-KDEL was progressively glycosylated in the endoplasmic reticulum (ER) and remained stably associated with this compartment. B-fragment with a nonfunctional KDEL sequence (B-Glyc-KDELGL) was glycosylated with about the same kinetics as B-Glyc-KDEL but localized at steady state to the Golgi apparatus. Morphological studies showed that B-Glyc-KDEL was delivered from the plasma membrane, via endosomes and the cisternae of the Golgi apparatus, to the ER. Moreover, the addition of a sulfation site allowed us to show that B-Glyc-KDEL on transit to the ER entered the Golgi apparatus through the trans-Golgi network. Transport of B-Glyc-KDEL to the ER was slowed down by nocodazole, indicating that microtubules are important for the retrograde pathway. Our results document the existence of a continuous pathway from the plasma membrane to the endoplasmic reticulum via the Golgi apparatus and show that a fully folded exogenous protein arriving in the endoplasmic reticulum via this pathway can undergo N-glycosylation.

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Section: Discussionsupporting

confidence: 82%

Section: Fig 4 Colocalization Of B-glyc-kdel and B-glyc-kdelgl Withsupporting

confidence: 81%

Retrograde Transport of KDEL-bearing B-fragment of Shiga Toxin

Johannes¹,

Tenza

Antony

et al. 1997

Journal of Biological Chemistry

187

254

View full text Add to dashboard Cite

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“…However, live cell imaging studies reveal that, for several minutes after entering the cell, endocytic probes in early ("sorting") endosomes remain fairly stationary and do not undergo long-range, microtubule-dependent translocations (Kim et al, 1996). Data were acquired from at least two different coverslips in two independent experiments.…”

Section: Association Of Dynactin and Clip-170 At Microtubule Endsmentioning

confidence: 99%

“…Endosomal pH was measured using an FITC pH ratio imaging technique (Kim et al, 1996). Cells overexpressing dynamitin were identified on the basis of Golgi morphology (Texas Red ceramide staining; Molecular Probes), loaded with FITC-␣ 2 -M (0.4 mg/ml) for 20 min at 37°C, then mounted in the heating stage of the microscope.…”

Section: Measurement Of Endosome Phmentioning

confidence: 99%

“…The fluorescence intensity (emission cutoff, 535 Ϯ 20 nm) of random fields was imaged with a slow scan charge-coupled device camera (VME200A; Photometrics, Tucson, AZ) and the 490:440 ratio was calculated (Ratiotool software; Inovision, Durham, NC). At the end of each experiment, a calibration curve of fluorescence intensity versus pH was generated in situ (Kim et al, 1996) and used to estimate the pH of individual fluorescent particles.…”

Section: Measurement Of Endosome Phmentioning

confidence: 99%

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Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170

Valetti

Wetzel

Schrader

et al. 1999

MBoC

256

252

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The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170’s putative cargo-binding domain. Overexpression studies using p150Glued, the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.

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Ammonium alters N-glycan structures of recombinant TNFR-IgG: Degradative versus biosynthetic mechanisms

Gawlitzek¹,

Ryll²,

Lofgren³

et al. 2000

Biotechnol. Bioeng.

161

149

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The effect of ammonium on the glycosylation pattern of the recombinant immunoadhesin tumor necrosis factor–IgG (TNFR‐IgG) produced by Chinese hamster ovary cells is elucidated in this study. TNFR‐IgG is a chimeric IgG fusion protein bearing one N‐linked glycosylation site in the Fc region and three complex‐type N‐glycans in the TNF‐receptor portion of each monomer. The ammonium concentration of batch suspension cultures was adjusted with glutamine and/or NH4Cl. The amount of galactose (Gal) and N‐acetylneuraminic acid (NANA) residues on TNFR‐IgG correlated in a dose‐dependent manner with the ammonium concentration under which the N‐linked oligosaccharides were synthesized. As ammonium increased from 1 to 15 mM, a concomitant decrease of up to 40% was observed in terminal galactosylation and sialylation of the molecule. Cell culture supernatants contained measurable β‐galactosidase and sialidase activity, which increased throughout the culture. The β‐galactosidase, but not the sialidase, level was proportional to the ammonium concentration. No loss of N‐glycans was observed in incubation studies using β‐galactosidase and sialidase containing cell culture supernatants, suggesting that the ammonium effect was biosynthetic and not degradative. Several biosynthetic mechanisms were investigated. Ammonium (a weak base) is known to affect the pH of acidic intracellular compartments (e.g., trans‐Golgi) as well as intracellular nucleotide sugar pools (increases UDP‐N‐acetylglucosamine and UDP‐N‐acetylgalactosamine). Ammonium might also affect the expression rates of β1,4‐galactosyltransferase (β1,4‐GT) and α2,3‐sialyltransferase (α2,3‐ST). To separate these mechanisms, experiments were designed using chloroquine (changes intracellular pH) and glucosamine (increases UDP‐GNAc pool [sum of UDP‐GlcNAc and UDP‐GalNAc]). The ammonium effect on TNFR‐IgG oligosaccharide structures could be mimicked only by chloroquine, another weak base. No differences in N‐glycosylation were found in the product synthesized in the presence of glucosamine. No differences in β1,4‐galactosyltransferase (β1,4‐GT) and α2,3‐sialyltransferase (α2,3‐ST) messenger RNA (mRNA) and enzyme levels were observed in cells cultivated in the presence or absence of 13 mM NH4Cl. pH titration of endogenous CHO α2,3‐ST and β‐1,4‐GT revealed a sharp optimum at pH 6.5, the reported trans‐Golgi pH. Thus, at pH 7.0 to 7.2, a likely trans‐Golgi pH range in the presence of 10 to 15 mM ammonium, activities for both enzymes are reduced to 50% to 60%. Consequently, ammonium seems to alter the carbohydrate biosynthesis of TNFR‐IgG by a pH‐mediated effect on glycosyltransferase activity. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 637–646, 2000.

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Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor.

Cited by 137 publications

References 38 publications

Retrograde Transport of KDEL-bearing B-fragment of Shiga Toxin

Retrograde Transport of KDEL-bearing B-fragment of Shiga Toxin

Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170

Ammonium alters N-glycan structures of recombinant TNFR-IgG: Degradative versus biosynthetic mechanisms

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