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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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Distribution of COP-I subunits in CHO and ldlF cells. Cytosols  were prepared from ldlF cells incubated at the permissive (34°C) or restrictive (40°C) temperature. For comparison, cytosols were also prepared  from WT CHO cells incubated at 37 or  40°C. The COP-I composition of each  cytosol was analyzed by SDS-PAGE  followed by Western blotting using antibodies against each of the COP-I components. 20 μg protein was loaded per  lane.
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Figure 1: Distribution of COP-I subunits in CHO and ldlF cells. Cytosols were prepared from ldlF cells incubated at the permissive (34°C) or restrictive (40°C) temperature. For comparison, cytosols were also prepared from WT CHO cells incubated at 37 or 40°C. The COP-I composition of each cytosol was analyzed by SDS-PAGE followed by Western blotting using antibodies against each of the COP-I components. 20 μg protein was loaded per lane.

Mentions: It has been previously shown that ts-εCOP is rapidly degraded in ldlF cells at the restrictive temperature (Guo et al., 1996) and that already at the permissive temperature, amounts of εCOP are reduced when compared with WT CHO cells (Guo et al., 1996; Fig. 1). The other COP subunits (β, β′, γ, δ, and ζCOP) were not affected by incubation at the restrictive temperature, except for an approximately twofold reduction in αCOP. Amounts of each subunit were, in fact, comparable to those found in WT CHO cells cultured at 37 or 40°C (Fig. 1), indicating that COP-I subunits or subcomplexes were stable despite the complete degradation of one subunit.


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

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

Distribution of COP-I subunits in CHO and ldlF cells. Cytosols  were prepared from ldlF cells incubated at the permissive (34°C) or restrictive (40°C) temperature. For comparison, cytosols were also prepared  from WT CHO cells incubated at 37 or  40°C. The COP-I composition of each  cytosol was analyzed by SDS-PAGE  followed by Western blotting using antibodies against each of the COP-I components. 20 μg protein was loaded per  lane.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Distribution of COP-I subunits in CHO and ldlF cells. Cytosols were prepared from ldlF cells incubated at the permissive (34°C) or restrictive (40°C) temperature. For comparison, cytosols were also prepared from WT CHO cells incubated at 37 or 40°C. The COP-I composition of each cytosol was analyzed by SDS-PAGE followed by Western blotting using antibodies against each of the COP-I components. 20 μg protein was loaded per lane.
Mentions: It has been previously shown that ts-εCOP is rapidly degraded in ldlF cells at the restrictive temperature (Guo et al., 1996) and that already at the permissive temperature, amounts of εCOP are reduced when compared with WT CHO cells (Guo et al., 1996; Fig. 1). The other COP subunits (β, β′, γ, δ, and ζCOP) were not affected by incubation at the restrictive temperature, except for an approximately twofold reduction in αCOP. Amounts of each subunit were, in fact, comparable to those found in WT CHO cells cultured at 37 or 40°C (Fig. 1), indicating that COP-I subunits or subcomplexes were stable despite the complete degradation of one subunit.

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