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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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Endocytosis in ldlF cells. Cells were incubated at the  permissive (34°C) or restrictive (40°C) temperature for 6 h. (A)  Transferrin internalization. Cells had been transiently transfected  with the cDNA encoding for the human transferrin receptor before incubation at 34 or 40°C. Transferrin internalization was visualized by fluorescence microscopy after 5 min incubation with  50 μg/ml rhodamine-transferrin at the corresponding temperature. (B) Continuous internalization of HRP. Cells were incubated with 3 mg/ml HRP at the corresponding temperature for 5,  15, 30, 60, or 120 min. The amounts of endocytosed HRP were  quantified and expressed as OD U/min/mg cellular protein. (C)  Recycling of internalized HRP. Cells were incubated with 0.5 mg/ ml HRP for 5 min at the corresponding temperature, washed, and  then reincubated for 10, 20, 30, or 40 min. At each time point,  cells and the media were collected. At each time point, HRP remaining associated to the monolayer is expressed as a percentage  of the total (cell-associated and regurgitated). In B and C, each  panel shows the mean of two representative series of experiments. Bar, 5 μm.
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Figure 2: Endocytosis in ldlF cells. Cells were incubated at the permissive (34°C) or restrictive (40°C) temperature for 6 h. (A) Transferrin internalization. Cells had been transiently transfected with the cDNA encoding for the human transferrin receptor before incubation at 34 or 40°C. Transferrin internalization was visualized by fluorescence microscopy after 5 min incubation with 50 μg/ml rhodamine-transferrin at the corresponding temperature. (B) Continuous internalization of HRP. Cells were incubated with 3 mg/ml HRP at the corresponding temperature for 5, 15, 30, 60, or 120 min. The amounts of endocytosed HRP were quantified and expressed as OD U/min/mg cellular protein. (C) Recycling of internalized HRP. Cells were incubated with 0.5 mg/ ml HRP for 5 min at the corresponding temperature, washed, and then reincubated for 10, 20, 30, or 40 min. At each time point, cells and the media were collected. At each time point, HRP remaining associated to the monolayer is expressed as a percentage of the total (cell-associated and regurgitated). In B and C, each panel shows the mean of two representative series of experiments. Bar, 5 μm.

Mentions: As a first step, we investigated whether early steps of the endocytic pathway were affected at the restrictive temperature. Cells were transiently transfected with the cDNA encoding for the human transferrin receptor (Zerial et al., 1987; Harder and Gerke, 1993), a well-established marker of clathrin-dependent endocytosis (Pearse and Robinson, 1990; Trowbridge et al., 1993) and then incubated at the desired temperature for 6 h. At this time, εCOP was fully degraded (Guo et al., 1996; data not shown), yet all other COP-I subunits were present (see Fig. 1). Then, the cells were incubated with human rhodamine-transferrin for only 5 min at the corresponding temperature, to allow one wave of receptor-mediated endocytosis to occur. As shown in Fig. 2 A, transferrin appeared to be internalized as efficiently at the restrictive or permissive temperature. Similarly CD4, which is endocytosed through clathrin-coated pits in T cell lines and transfected HeLa cells, was internalized at similar rates in stably transfected ldlF cells incubated at permissive or restrictive temperature (Bowers, K., and M. Marsh personal communication). These data indicate that clathrin-dependent endocytosis continued in the absence 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)

Endocytosis in ldlF cells. Cells were incubated at the  permissive (34°C) or restrictive (40°C) temperature for 6 h. (A)  Transferrin internalization. Cells had been transiently transfected  with the cDNA encoding for the human transferrin receptor before incubation at 34 or 40°C. Transferrin internalization was visualized by fluorescence microscopy after 5 min incubation with  50 μg/ml rhodamine-transferrin at the corresponding temperature. (B) Continuous internalization of HRP. Cells were incubated with 3 mg/ml HRP at the corresponding temperature for 5,  15, 30, 60, or 120 min. The amounts of endocytosed HRP were  quantified and expressed as OD U/min/mg cellular protein. (C)  Recycling of internalized HRP. Cells were incubated with 0.5 mg/ ml HRP for 5 min at the corresponding temperature, washed, and  then reincubated for 10, 20, 30, or 40 min. At each time point,  cells and the media were collected. At each time point, HRP remaining associated to the monolayer is expressed as a percentage  of the total (cell-associated and regurgitated). In B and C, each  panel shows the mean of two representative series of experiments. Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 2: Endocytosis in ldlF cells. Cells were incubated at the permissive (34°C) or restrictive (40°C) temperature for 6 h. (A) Transferrin internalization. Cells had been transiently transfected with the cDNA encoding for the human transferrin receptor before incubation at 34 or 40°C. Transferrin internalization was visualized by fluorescence microscopy after 5 min incubation with 50 μg/ml rhodamine-transferrin at the corresponding temperature. (B) Continuous internalization of HRP. Cells were incubated with 3 mg/ml HRP at the corresponding temperature for 5, 15, 30, 60, or 120 min. The amounts of endocytosed HRP were quantified and expressed as OD U/min/mg cellular protein. (C) Recycling of internalized HRP. Cells were incubated with 0.5 mg/ ml HRP for 5 min at the corresponding temperature, washed, and then reincubated for 10, 20, 30, or 40 min. At each time point, cells and the media were collected. At each time point, HRP remaining associated to the monolayer is expressed as a percentage of the total (cell-associated and regurgitated). In B and C, each panel shows the mean of two representative series of experiments. Bar, 5 μm.
Mentions: As a first step, we investigated whether early steps of the endocytic pathway were affected at the restrictive temperature. Cells were transiently transfected with the cDNA encoding for the human transferrin receptor (Zerial et al., 1987; Harder and Gerke, 1993), a well-established marker of clathrin-dependent endocytosis (Pearse and Robinson, 1990; Trowbridge et al., 1993) and then incubated at the desired temperature for 6 h. At this time, εCOP was fully degraded (Guo et al., 1996; data not shown), yet all other COP-I subunits were present (see Fig. 1). Then, the cells were incubated with human rhodamine-transferrin for only 5 min at the corresponding temperature, to allow one wave of receptor-mediated endocytosis to occur. As shown in Fig. 2 A, transferrin appeared to be internalized as efficiently at the restrictive or permissive temperature. Similarly CD4, which is endocytosed through clathrin-coated pits in T cell lines and transfected HeLa cells, was internalized at similar rates in stably transfected ldlF cells incubated at permissive or restrictive temperature (Bowers, K., and M. Marsh personal communication). These data indicate that clathrin-dependent endocytosis continued in the absence of εCOP.

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