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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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Ultrastructure of ldlF endosomes at the permissive  temperature. Cells cultured at 34°C were incubated with HRP for  5 min to label early endosomes and then either fixed immediately  (A and B) or further incubated at 34°C for 30 min to label late endosomes (C). Cells were then processed for plastic sections, and  semithick (∼150 nm; A and C) or ultrathin sections (50 nm; B)  were prepared. Early endosomal compartments (A and B) are  comprised of tubular and cisternal regions (arrows) and vesicular  domains (arrowheads), as in other cells. B shows a higher magnification view of the Golgi (g) area. After further incubation for 30  min (C), HRP was rarely observed within tubular domains. As  expected, it was distributed within larger multivesicular elements  concentrated in the Golgi area (arrowheads), which presumably  correspond to late endosomes. Bars, 0.5 μm.
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Figure 7: Ultrastructure of ldlF endosomes at the permissive temperature. Cells cultured at 34°C were incubated with HRP for 5 min to label early endosomes and then either fixed immediately (A and B) or further incubated at 34°C for 30 min to label late endosomes (C). Cells were then processed for plastic sections, and semithick (∼150 nm; A and C) or ultrathin sections (50 nm; B) were prepared. Early endosomal compartments (A and B) are comprised of tubular and cisternal regions (arrows) and vesicular domains (arrowheads), as in other cells. B shows a higher magnification view of the Golgi (g) area. After further incubation for 30 min (C), HRP was rarely observed within tubular domains. As expected, it was distributed within larger multivesicular elements concentrated in the Golgi area (arrowheads), which presumably correspond to late endosomes. Bars, 0.5 μm.

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)

Ultrastructure of ldlF endosomes at the permissive  temperature. Cells cultured at 34°C were incubated with HRP for  5 min to label early endosomes and then either fixed immediately  (A and B) or further incubated at 34°C for 30 min to label late endosomes (C). Cells were then processed for plastic sections, and  semithick (∼150 nm; A and C) or ultrathin sections (50 nm; B)  were prepared. Early endosomal compartments (A and B) are  comprised of tubular and cisternal regions (arrows) and vesicular  domains (arrowheads), as in other cells. B shows a higher magnification view of the Golgi (g) area. After further incubation for 30  min (C), HRP was rarely observed within tubular domains. As  expected, it was distributed within larger multivesicular elements  concentrated in the Golgi area (arrowheads), which presumably  correspond to late endosomes. Bars, 0.5 μm.
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Related In: Results  -  Collection

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Figure 7: Ultrastructure of ldlF endosomes at the permissive temperature. Cells cultured at 34°C were incubated with HRP for 5 min to label early endosomes and then either fixed immediately (A and B) or further incubated at 34°C for 30 min to label late endosomes (C). Cells were then processed for plastic sections, and semithick (∼150 nm; A and C) or ultrathin sections (50 nm; B) were prepared. Early endosomal compartments (A and B) are comprised of tubular and cisternal regions (arrows) and vesicular domains (arrowheads), as in other cells. B shows a higher magnification view of the Golgi (g) area. After further incubation for 30 min (C), HRP was rarely observed within tubular domains. As expected, it was distributed within larger multivesicular elements concentrated in the Golgi area (arrowheads), which presumably correspond to late endosomes. Bars, 0.5 μm.
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