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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 different markers in ldlF cells. (A) Cells  prepared as in Fig. 3 were processed for immunofluorescence microscopy using human antiserum against EEA1. Arrows point at  changes in the appearance of early endosomal elements at 40°C,  when compared with 34°C. low, low magnification; high, high magnification. (B) Cells were processed for immunofluorescence microscopy as in A using the maD antibody against βCOP or a rabbit antiserum against either the Man6P-R, rab7, or calnexin. The  maD antibody does not label endosomal βCOP to any significant  extent under these conditions. Bars, 5 μm.
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Figure 6: Distribution of different markers in ldlF cells. (A) Cells prepared as in Fig. 3 were processed for immunofluorescence microscopy using human antiserum against EEA1. Arrows point at changes in the appearance of early endosomal elements at 40°C, when compared with 34°C. low, low magnification; high, high magnification. (B) Cells were processed for immunofluorescence microscopy as in A using the maD antibody against βCOP or a rabbit antiserum against either the Man6P-R, rab7, or calnexin. The maD antibody does not label endosomal βCOP to any significant extent under these conditions. Bars, 5 μm.

Mentions: We then investigated whether the organization of endosomes was affected in the absence of εCOP. First, we used antibodies against EEA1, a recently discovered protein that localizes to early endosomal vesicles (Mu et al., 1994). At the permissive temperature, EEA1-positive elements exhibited a highly punctate distribution (Fig. 6 A), which is characteristic for this protein. Although the overall topology was similar at the restrictive temperatures, EEA1- labeled elements then appeared more clustered or tubular even at the light microscopy level, as illustrated in higher magnification images (Fig. 6 A). In contrast, the distribution of a late endosomal marker, the small GTPase rab7 (Chavrier et al., 1990), remained unchanged after incubation at the restrictive temperature for 6 h (Fig. 6 B), as was the distribution of the cation-independent mannose-6-phosphate receptor, which localizes primarily to late endosomes and TGN (Brown et al., 1986; Griffiths et al., 1988), and calnexin, a marker of the endoplasmic reticulum (Wada et al., 1991). As reported by Guo et al. (1994), we also observed some redistribution of βCOP at the restrictive temperature, using an antibody that recognizes primarily biosynthetic βCOP by immunofluorescence.


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 different markers in ldlF cells. (A) Cells  prepared as in Fig. 3 were processed for immunofluorescence microscopy using human antiserum against EEA1. Arrows point at  changes in the appearance of early endosomal elements at 40°C,  when compared with 34°C. low, low magnification; high, high magnification. (B) Cells were processed for immunofluorescence microscopy as in A using the maD antibody against βCOP or a rabbit antiserum against either the Man6P-R, rab7, or calnexin. The  maD antibody does not label endosomal βCOP to any significant  extent under these conditions. Bars, 5 μm.
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Related In: Results  -  Collection

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

Figure 6: Distribution of different markers in ldlF cells. (A) Cells prepared as in Fig. 3 were processed for immunofluorescence microscopy using human antiserum against EEA1. Arrows point at changes in the appearance of early endosomal elements at 40°C, when compared with 34°C. low, low magnification; high, high magnification. (B) Cells were processed for immunofluorescence microscopy as in A using the maD antibody against βCOP or a rabbit antiserum against either the Man6P-R, rab7, or calnexin. The maD antibody does not label endosomal βCOP to any significant extent under these conditions. Bars, 5 μm.
Mentions: We then investigated whether the organization of endosomes was affected in the absence of εCOP. First, we used antibodies against EEA1, a recently discovered protein that localizes to early endosomal vesicles (Mu et al., 1994). At the permissive temperature, EEA1-positive elements exhibited a highly punctate distribution (Fig. 6 A), which is characteristic for this protein. Although the overall topology was similar at the restrictive temperatures, EEA1- labeled elements then appeared more clustered or tubular even at the light microscopy level, as illustrated in higher magnification images (Fig. 6 A). In contrast, the distribution of a late endosomal marker, the small GTPase rab7 (Chavrier et al., 1990), remained unchanged after incubation at the restrictive temperature for 6 h (Fig. 6 B), as was the distribution of the cation-independent mannose-6-phosphate receptor, which localizes primarily to late endosomes and TGN (Brown et al., 1986; Griffiths et al., 1988), and calnexin, a marker of the endoplasmic reticulum (Wada et al., 1991). As reported by Guo et al. (1994), we also observed some redistribution of βCOP at the restrictive temperature, using an antibody that recognizes primarily biosynthetic βCOP by immunofluorescence.

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