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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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Subcellular fractionation of ldlF endosomes.  (A) Cells prepared as in Fig.  3 were incubated at the permissive (34°C) or restrictive  (40°C) temperature with medium containing 4 mg/ml  HRP for 5 min (pulse) or  subsequently reincubated  for 40 min in the absence of  marker (chase). Early (EE)  and late (LE) endosomes  were then separated by subcellular fractionation. The total HRP content of the fractions was quantified using a  colorimetric reaction and is  expressed as OD U/min.  HRP recovery in the fractions corresponded to ∼40%  of the total, latent cell-associated activity (∼50% lost to  the nuclear pellet after gentle  homogenization) and HRP  latency after homogenization  was always >70%. (B) The recovery of rab5 and rab7 in fractions  (EE or LE at each temperature) prepared as in A was analyzed  by SDS gel electrophoresis, followed by Western blotting with indicated antibodies. Each lane contained 20% of the total protein  in the corresponding fraction.
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Figure 4: Subcellular fractionation of ldlF endosomes. (A) Cells prepared as in Fig. 3 were incubated at the permissive (34°C) or restrictive (40°C) temperature with medium containing 4 mg/ml HRP for 5 min (pulse) or subsequently reincubated for 40 min in the absence of marker (chase). Early (EE) and late (LE) endosomes were then separated by subcellular fractionation. The total HRP content of the fractions was quantified using a colorimetric reaction and is expressed as OD U/min. HRP recovery in the fractions corresponded to ∼40% of the total, latent cell-associated activity (∼50% lost to the nuclear pellet after gentle homogenization) and HRP latency after homogenization was always >70%. (B) The recovery of rab5 and rab7 in fractions (EE or LE at each temperature) prepared as in A was analyzed by SDS gel electrophoresis, followed by Western blotting with indicated antibodies. Each lane contained 20% of the total protein in the corresponding fraction.

Mentions: Our previous data showed that βCOP is present on early endosomes and is required for the formation of vesicles that mediate transport from early to late endosomes in vitro (Aniento et al., 1996). We therefore investigated whether early to late endosome transport still occurred in ldlF cells incubated at the restrictive temperature for 6 h in vivo. Early endosomes were labeled with HRP internalized for 5 min from the medium. To label late endosomes, HRP was subsequently chased for 30 min in marker-free medium (Gruenberg and Howell, 1989; Aniento et al., 1993). The subcellular distribution of HRP was analyzed by subcellular fractionation (Fig. 4), as well as by light (Fig. 3) and electron (Figs. 7 and 8) microscopy.


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

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

Subcellular fractionation of ldlF endosomes.  (A) Cells prepared as in Fig.  3 were incubated at the permissive (34°C) or restrictive  (40°C) temperature with medium containing 4 mg/ml  HRP for 5 min (pulse) or  subsequently reincubated  for 40 min in the absence of  marker (chase). Early (EE)  and late (LE) endosomes  were then separated by subcellular fractionation. The total HRP content of the fractions was quantified using a  colorimetric reaction and is  expressed as OD U/min.  HRP recovery in the fractions corresponded to ∼40%  of the total, latent cell-associated activity (∼50% lost to  the nuclear pellet after gentle  homogenization) and HRP  latency after homogenization  was always >70%. (B) The recovery of rab5 and rab7 in fractions  (EE or LE at each temperature) prepared as in A was analyzed  by SDS gel electrophoresis, followed by Western blotting with indicated antibodies. Each lane contained 20% of the total protein  in the corresponding fraction.
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Related In: Results  -  Collection

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Figure 4: Subcellular fractionation of ldlF endosomes. (A) Cells prepared as in Fig. 3 were incubated at the permissive (34°C) or restrictive (40°C) temperature with medium containing 4 mg/ml HRP for 5 min (pulse) or subsequently reincubated for 40 min in the absence of marker (chase). Early (EE) and late (LE) endosomes were then separated by subcellular fractionation. The total HRP content of the fractions was quantified using a colorimetric reaction and is expressed as OD U/min. HRP recovery in the fractions corresponded to ∼40% of the total, latent cell-associated activity (∼50% lost to the nuclear pellet after gentle homogenization) and HRP latency after homogenization was always >70%. (B) The recovery of rab5 and rab7 in fractions (EE or LE at each temperature) prepared as in A was analyzed by SDS gel electrophoresis, followed by Western blotting with indicated antibodies. Each lane contained 20% of the total protein in the corresponding fraction.
Mentions: Our previous data showed that βCOP is present on early endosomes and is required for the formation of vesicles that mediate transport from early to late endosomes in vitro (Aniento et al., 1996). We therefore investigated whether early to late endosome transport still occurred in ldlF cells incubated at the restrictive temperature for 6 h in vivo. Early endosomes were labeled with HRP internalized for 5 min from the medium. To label late endosomes, HRP was subsequently chased for 30 min in marker-free medium (Gruenberg and Howell, 1989; Aniento et al., 1993). The subcellular distribution of HRP was analyzed by subcellular fractionation (Fig. 4), as well as by light (Fig. 3) and electron (Figs. 7 and 8) microscopy.

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