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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.However, alphaCOP, which is normally present on endosomes, is no longer recruited when epsilonCOP is missing.Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins.

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 restrictive temperature. Cells  cultured at 40°C for 6 h were  incubated with HRP for 5  min and then either fixed immediately (A and B) or further incubated at 40°C for 30  min (C–E). Semithick (A and  C) or ultrathin (B, D, and E)  sections of the cell pellet  were prepared as in Fig. 8.  Early endosomal compartments (A and B) were composed of small tubular and  vesicular elements that were  predominantly in discrete  clusters (arrows). Few large  vesicular profiles were evident (compare with Fig. 7, A  and B). After further incubation for 30 min, little HRP remained in the cells (see Figs.  2–4). However, when detected (C–E), the bulk of  HRP was still observed  within clusters of tubular and  vesicular elements (arrows),  which appeared identical to  those labeled after the 10  min pulse. Few vesicular elements were labeled (arrowhead). Note the clear difference when compared with  cells incubated for the same  time at the permissive temperature (Fig. 7 C). As  shown at higher magnification (D and E), labeled elements comprise vesicles and  short tubules, which appear  discontinuous from the analysis of both semithick (C)  and thin (D and E) sections.  Bars, 0.5 μm.
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Figure 8: Ultrastructure of ldlF endosomes at the restrictive temperature. Cells cultured at 40°C for 6 h were incubated with HRP for 5 min and then either fixed immediately (A and B) or further incubated at 40°C for 30 min (C–E). Semithick (A and C) or ultrathin (B, D, and E) sections of the cell pellet were prepared as in Fig. 8. Early endosomal compartments (A and B) were composed of small tubular and vesicular elements that were predominantly in discrete clusters (arrows). Few large vesicular profiles were evident (compare with Fig. 7, A and B). After further incubation for 30 min, little HRP remained in the cells (see Figs. 2–4). However, when detected (C–E), the bulk of HRP was still observed within clusters of tubular and vesicular elements (arrows), which appeared identical to those labeled after the 10 min pulse. Few vesicular elements were labeled (arrowhead). Note the clear difference when compared with cells incubated for the same time at the permissive temperature (Fig. 7 C). As shown at higher magnification (D and E), labeled elements comprise vesicles and short tubules, which appear discontinuous from the analysis of both semithick (C) and thin (D and E) sections. 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 restrictive temperature. Cells  cultured at 40°C for 6 h were  incubated with HRP for 5  min and then either fixed immediately (A and B) or further incubated at 40°C for 30  min (C–E). Semithick (A and  C) or ultrathin (B, D, and E)  sections of the cell pellet  were prepared as in Fig. 8.  Early endosomal compartments (A and B) were composed of small tubular and  vesicular elements that were  predominantly in discrete  clusters (arrows). Few large  vesicular profiles were evident (compare with Fig. 7, A  and B). After further incubation for 30 min, little HRP remained in the cells (see Figs.  2–4). However, when detected (C–E), the bulk of  HRP was still observed  within clusters of tubular and  vesicular elements (arrows),  which appeared identical to  those labeled after the 10  min pulse. Few vesicular elements were labeled (arrowhead). Note the clear difference when compared with  cells incubated for the same  time at the permissive temperature (Fig. 7 C). As  shown at higher magnification (D and E), labeled elements comprise vesicles and  short tubules, which appear  discontinuous from the analysis of both semithick (C)  and thin (D and E) sections.  Bars, 0.5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140201&req=5

Figure 8: Ultrastructure of ldlF endosomes at the restrictive temperature. Cells cultured at 40°C for 6 h were incubated with HRP for 5 min and then either fixed immediately (A and B) or further incubated at 40°C for 30 min (C–E). Semithick (A and C) or ultrathin (B, D, and E) sections of the cell pellet were prepared as in Fig. 8. Early endosomal compartments (A and B) were composed of small tubular and vesicular elements that were predominantly in discrete clusters (arrows). Few large vesicular profiles were evident (compare with Fig. 7, A and B). After further incubation for 30 min, little HRP remained in the cells (see Figs. 2–4). However, when detected (C–E), the bulk of HRP was still observed within clusters of tubular and vesicular elements (arrows), which appeared identical to those labeled after the 10 min pulse. Few vesicular elements were labeled (arrowhead). Note the clear difference when compared with cells incubated for the same time at the permissive temperature (Fig. 7 C). As shown at higher magnification (D and E), labeled elements comprise vesicles and short tubules, which appear discontinuous from the analysis of both semithick (C) and thin (D and E) sections. 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.However, alphaCOP, which is normally present on endosomes, is no longer recruited when epsilonCOP is missing.Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins.

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