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Functional dissection of COP-I subunits in the biogenesis of multivesicular endosomes.

Gu F, Aniento F, Parton RG, Gruenberg J - J. Cell Biol. (1997)

Bottom Line: Previous studies showed that gamma and deltaCOP are not found on endosomes.Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins.Our data also show that membrane association and function of endosomal COPs can be dissected: whereas beta, beta', and zetaCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of alpha and/or epsilonCOP.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, University of Geneva, 1211-Geneva-4, Switzerland.

ABSTRACT
In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in epsilon coatomer protein (epsilonCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although epsilonCOP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of epsilonCOP, we find that a subcomplex of beta, beta', and zetaCOP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPgammaS and sensitivity to the endosomal pH. Previous studies showed that gamma and deltaCOP are not found on endosomes. However, alphaCOP, which is normally present on endosomes, is no longer recruited when epsilonCOP is missing. In contrast, all COP subunits, except obviously epsilonCOP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas beta, beta', and zetaCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of alpha and/or epsilonCOP.

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Acidification in ldlF cells. Cells prepared as in Fig. 3 at  the permissive or restrictive temperature were treated with acridine orange (A.O.), LysoSensor acidic (detection range pH 4.5–6;  LS. a) and LysoSensor neutral (detection range pH 6.5–8; LS. n)  for 10 min to reveal acidic compartments. The intrinsic fluorescence of each dye after accumulation within acidic endosomes and  lysosomes was observed by fluorescence microscopy. Bar, 5 μm.
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Figure 5: Acidification in ldlF cells. Cells prepared as in Fig. 3 at the permissive or restrictive temperature were treated with acridine orange (A.O.), LysoSensor acidic (detection range pH 4.5–6; LS. a) and LysoSensor neutral (detection range pH 6.5–8; LS. n) for 10 min to reveal acidic compartments. The intrinsic fluorescence of each dye after accumulation within acidic endosomes and lysosomes was observed by fluorescence microscopy. Bar, 5 μm.

Mentions: In our previous studies, we had observed that neutralization of the endosomal pH also caused inhibition of early to late endosome transport (Aniento et al., 1996; Clague et al., 1994). We thus investigated whether ldlF cells exhibited an acidification defect at the restrictive temperature, using the pH-sensitive dyes acridine orange and two forms of LysoSensor, which detect pH values in the 4.5– 6.0 and 6.5–8.0 ranges, respectively. As shown in Fig. 5, no difference could be observed between cells incubated at the permissive or restrictive temperature for 6 h. In fact, we did not observe any difference with anyone of the three dyes when cells were incubated at permissive or restrictive temperature over a time period ranging from 1 to 12 h (not shown). These experiments show that, after εCOP degradation, major differences in the acidification properties of endosomes and lysosomes could not be detected using these dyes. Altogether, our biochemical and morphological data indicate that transport from early to late endosomes is inhibited in vivo, at the restrictive temperature.


Functional dissection of COP-I subunits in the biogenesis of multivesicular endosomes.

Gu F, Aniento F, Parton RG, Gruenberg J - J. Cell Biol. (1997)

Acidification in ldlF cells. Cells prepared as in Fig. 3 at  the permissive or restrictive temperature were treated with acridine orange (A.O.), LysoSensor acidic (detection range pH 4.5–6;  LS. a) and LysoSensor neutral (detection range pH 6.5–8; LS. n)  for 10 min to reveal acidic compartments. The intrinsic fluorescence of each dye after accumulation within acidic endosomes and  lysosomes was observed by fluorescence microscopy. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140201&req=5

Figure 5: Acidification in ldlF cells. Cells prepared as in Fig. 3 at the permissive or restrictive temperature were treated with acridine orange (A.O.), LysoSensor acidic (detection range pH 4.5–6; LS. a) and LysoSensor neutral (detection range pH 6.5–8; LS. n) for 10 min to reveal acidic compartments. The intrinsic fluorescence of each dye after accumulation within acidic endosomes and lysosomes was observed by fluorescence microscopy. Bar, 5 μm.
Mentions: In our previous studies, we had observed that neutralization of the endosomal pH also caused inhibition of early to late endosome transport (Aniento et al., 1996; Clague et al., 1994). We thus investigated whether ldlF cells exhibited an acidification defect at the restrictive temperature, using the pH-sensitive dyes acridine orange and two forms of LysoSensor, which detect pH values in the 4.5– 6.0 and 6.5–8.0 ranges, respectively. As shown in Fig. 5, no difference could be observed between cells incubated at the permissive or restrictive temperature for 6 h. In fact, we did not observe any difference with anyone of the three dyes when cells were incubated at permissive or restrictive temperature over a time period ranging from 1 to 12 h (not shown). These experiments show that, after εCOP degradation, major differences in the acidification properties of endosomes and lysosomes could not be detected using these dyes. Altogether, our biochemical and morphological data indicate that transport from early to late endosomes is inhibited in vivo, at the restrictive temperature.

Bottom Line: Previous studies showed that gamma and deltaCOP are not found on endosomes.Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins.Our data also show that membrane association and function of endosomal COPs can be dissected: whereas beta, beta', and zetaCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of alpha and/or epsilonCOP.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, University of Geneva, 1211-Geneva-4, Switzerland.

ABSTRACT
In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in epsilon coatomer protein (epsilonCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although epsilonCOP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of epsilonCOP, we find that a subcomplex of beta, beta', and zetaCOP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPgammaS and sensitivity to the endosomal pH. Previous studies showed that gamma and deltaCOP are not found on endosomes. However, alphaCOP, which is normally present on endosomes, is no longer recruited when epsilonCOP is missing. In contrast, all COP subunits, except obviously epsilonCOP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas beta, beta', and zetaCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of alpha and/or epsilonCOP.

Show MeSH
Related in: MedlinePlus