Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells

Kao, Chia Yi; Draper, Rockford K.

doi:10.1083/jcb.117.4.701

Cited by 21 publications

(18 citation statements)

References 59 publications

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“…80,81 Those are extreme examples, but they serve to illustrate that the identity of organelles is largely determined by the rates of entry and exit of membrane and cargo. It is reasonable to speculate that signalling as has been discussed here is used to maintain coordinated transport rates throughout the endomembrane system, thus contributing to the stability of the organelles themselves.…”

Section: Is One Point Of Signalling To Monitor and Regulate Recyclingmentioning

confidence: 98%

Signalling molecules and the regulation of intracellular transport

Ktistakis

1998

Bioessays

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Section: Is One Point Of Signalling To Monitor and Regulate Recyclingmentioning

confidence: 98%

Signalling molecules and the regulation of intracellular transport

Ktistakis

1998

Bioessays

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“…To examine the potential role of the Golgi complex as an intermediate compartment in the trafficking of LT, we employed the temperature-sensitive, mutant V.24.1 cell line. This cell line, belonging to the End4 complementation group of CH0-K1 cells, possesses a lesion that results in loss of Golgi structure and function at 41 C (Kao and Draper, 1992). Preliminary experiments revealed, as reported by Kao and Draper (1992) and Bau and Draper (1993), that V.24,1 cells exhibited a marked loss of Golgi structure after 2.5 h at 41 C, as judged by staining with the fluorescent ceramide analogue, Cs-NBD-ceramide, and that the lesion is maintained during subsequent incubation at r 1995 Blacicwell Science Ltd, Molecular Microbioiogy, 16, 789-800 CH0-K1 and V.24.1 cells were cultured in DMEM containing 1% FBS and 1 mM IBMX at either 34 C or 41 C for 2.5 h. The cells were then maintained at 34 C or shitted to 39.5 C prior to the addition of toxtn.…”

Section: Effect Of Lt On End4 Mutant V241 Cellsmentioning

confidence: 99%

Role of a potential endoplasmic reticulum retention sequence (RDEL) and the Golgi complex in the cytotonic activity of Escherichia coli heat‐labile enterotoxin

Cieplak

Messer

Konkel

et al. 1995

Molecular Microbiology

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Recent experimental evidence indicates that Escherichia coli heat-labile enterotoxin and the closely related cholera toxin gain access to intracellular target substrates through a brefeldin A-sensitive pathway that may involve retrograde transport through the Golgi-endoplasmic reticulum network. The A subunits of both toxins possess a carboxy-terminal tetrapeptide sequence (KDEL in cholera toxin and RDEL in the heat-labile enterotoxins) that is known to mediate the retention of eukaryotic proteins in the endoplasmic reticulum. To investigate the potential role of the RDEL sequence in the toxic activity of the heat-labile enterotoxin we constructed mutant analogues of the toxin containing single substitutions (RDGL and RDEV) or a reversed sequence (LEDR). The single substitutions had little effect on Chinese hamster ovary cell elongation or the ability to stimulate cAMP accumulation in Caco-2 cells. Reversal of the sequence reduced the ability of the toxin to increase cAMP levels in Caco-2 cells by approximately 60% and decreased the ability to elicit elongation of Chinese hamster ovary cells. The effects of the heat-labile enterotoxin were not diminished in a mutant Chinese hamster ovary cell line (V.24.1) that belongs to the End4 complementation group and possesses a temperature-sensitive block in secretion that correlates directly with the disappearance of the Golgi stacks. Collectively, these findings suggest that the brefeldin A-sensitive process involved in intoxication by the heat-labile enterotoxin does not involve RDEL-dependent retrograde transport of the A subunit through the Golgi-endoplasmic reticulum complex. The results are more consistent with a model of internalization involving translocation of the A subunit from an endosomal or a trans-Golgi network compartment.

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“…In addition, because in mammalian cells the morphology of the secretory organelles is better visualized and described, questions involving organelle identity and biogenesis may be more easily answered there. As an outstanding example of this, three mutant CHO cell lines have been isolated in which the onset of a block in secretion is accompanied by a rapid and reversible loss of the Golgi complex (Zuber et al, 1991;Kao and Draper, 1992;Guo et al, 1994). This phenotype, which is reminiscent of the effects of BFA on secretion and on the morphology of the Golgi complex, strongly suggests a coupling of secretory activity and organelle identity (Hurtley, 1992).…”

Section: Isolation Of Cho Cells With Defects In Secretionmentioning

confidence: 99%

A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

Ktistakis

Kao

Wang

et al. 1995

MBoC

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The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescenceactivated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10-6. Thus, the fluorescent lipid C5-DMBceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.

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Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells

Abstract: Abstract. Mutant V.24.1, a member of the End4 complementation group of temperature-sensitive CHO cells, is defective in secretion at the restrictive temperature

Cited by 21 publications

References 59 publications

Signalling molecules and the regulation of intracellular transport

Signalling molecules and the regulation of intracellular transport

Role of a potential endoplasmic reticulum retention sequence (RDEL) and the Golgi complex in the cytotonic activity of Escherichia coli heat‐labile enterotoxin

A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

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