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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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COP binding to  endosomal and biosynthetic  membranes. Cytosols were  prepared from ldlF cells incubated at the restrictive  temperature, as in Fig. 1.  The COP binding capacity  of biosynthetic (BM) or early  endosomal (EE) membranes  prepared from BHK cells  pretreated with brefeldin A  was tested as in Fig 10, except that ldlF cytosol was  used with (G and NG) or  without (C) 10 μM GTPγS.  When indicated (NG), the  pH of endosomes was preneutralized with 50 μM nigericin before GTPγS addition. Membranes were  retrieved and analyzed using  antibodies against each COP  subunit. Western blots were developed using the ECL reaction;  exposure times were five times longer for EE than for BM membranes to ensure that signals remained in the linear detection  range.
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Figure 11: COP binding to endosomal and biosynthetic membranes. Cytosols were prepared from ldlF cells incubated at the restrictive temperature, as in Fig. 1. The COP binding capacity of biosynthetic (BM) or early endosomal (EE) membranes prepared from BHK cells pretreated with brefeldin A was tested as in Fig 10, except that ldlF cytosol was used with (G and NG) or without (C) 10 μM GTPγS. When indicated (NG), the pH of endosomes was preneutralized with 50 μM nigericin before GTPγS addition. Membranes were retrieved and analyzed using antibodies against each COP subunit. Western blots were developed using the ECL reaction; exposure times were five times longer for EE than for BM membranes to ensure that signals remained in the linear detection range.

Mentions: To our surprise, we observed that COP binding onto endosomal membranes still occurred when εCOP was missing (Fig. 11; EE, early endosome fraction). However, a small COP subset only, consisting of β′, β, and ζCOP, was then recruited onto early endosomes. Previous experiments had shown that γ and δCOP are not present on endosomes (Whitney et al., 1995; Aniento et al., 1996), but in the absence of εCOP, αCOP also failed to interact with endosomes. In contrast, the same cytosol preparation was fully competent to support binding of all subunits, including α, γ, and δCOP, but obviously excluding εCOP, to biosynthetic membranes (Fig. 11; BM, biosynthetic membrane fraction). These experiments demonstrate that some COP subunits, namely β, β′, and ζCOP, are still recruited onto endosomal membranes despite the absence of εCOP. Since this subcomplex did not support membrane transport in vivo (Figs. 3, 4, 7, and 8) or efficient ECV/MVB formation in vitro (Fig. 9), our data suggest that COP function in endosome transport requires the presence of α and/or εCOP. Our data also indicate that recruitment of αCOP onto endosomal membranes, but not biosynthetic membranes, requires the presence of εCOP.


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

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

COP binding to  endosomal and biosynthetic  membranes. Cytosols were  prepared from ldlF cells incubated at the restrictive  temperature, as in Fig. 1.  The COP binding capacity  of biosynthetic (BM) or early  endosomal (EE) membranes  prepared from BHK cells  pretreated with brefeldin A  was tested as in Fig 10, except that ldlF cytosol was  used with (G and NG) or  without (C) 10 μM GTPγS.  When indicated (NG), the  pH of endosomes was preneutralized with 50 μM nigericin before GTPγS addition. Membranes were  retrieved and analyzed using  antibodies against each COP  subunit. Western blots were developed using the ECL reaction;  exposure times were five times longer for EE than for BM membranes to ensure that signals remained in the linear detection  range.
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Related In: Results  -  Collection

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Figure 11: COP binding to endosomal and biosynthetic membranes. Cytosols were prepared from ldlF cells incubated at the restrictive temperature, as in Fig. 1. The COP binding capacity of biosynthetic (BM) or early endosomal (EE) membranes prepared from BHK cells pretreated with brefeldin A was tested as in Fig 10, except that ldlF cytosol was used with (G and NG) or without (C) 10 μM GTPγS. When indicated (NG), the pH of endosomes was preneutralized with 50 μM nigericin before GTPγS addition. Membranes were retrieved and analyzed using antibodies against each COP subunit. Western blots were developed using the ECL reaction; exposure times were five times longer for EE than for BM membranes to ensure that signals remained in the linear detection range.
Mentions: To our surprise, we observed that COP binding onto endosomal membranes still occurred when εCOP was missing (Fig. 11; EE, early endosome fraction). However, a small COP subset only, consisting of β′, β, and ζCOP, was then recruited onto early endosomes. Previous experiments had shown that γ and δCOP are not present on endosomes (Whitney et al., 1995; Aniento et al., 1996), but in the absence of εCOP, αCOP also failed to interact with endosomes. In contrast, the same cytosol preparation was fully competent to support binding of all subunits, including α, γ, and δCOP, but obviously excluding εCOP, to biosynthetic membranes (Fig. 11; BM, biosynthetic membrane fraction). These experiments demonstrate that some COP subunits, namely β, β′, and ζCOP, are still recruited onto endosomal membranes despite the absence of εCOP. Since this subcomplex did not support membrane transport in vivo (Figs. 3, 4, 7, and 8) or efficient ECV/MVB formation in vitro (Fig. 9), our data suggest that COP function in endosome transport requires the presence of α and/or εCOP. Our data also indicate that recruitment of αCOP onto endosomal membranes, but not biosynthetic membranes, requires the presence of εCOP.

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