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Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins.

Paladino S, Sarnataro D, Pillich R, Tivodar S, Nitsch L, Zurzolo C - J. Cell Biol. (2004)

Bottom Line: Impairment of oligomerization leads to protein missorting.We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs.Two alternative apical sorting models are presented.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia e Patologia Cellulare e Molecolare, Centro di Endocrinologia ed Oncologia Sperimentale, CNR, Università degli Studi di Napoli Federico II, Italy.

ABSTRACT
An essential but insufficient step for apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in epithelial cells is their association with detergent-resistant microdomains (DRMs) or rafts. In this paper, we show that in MDCK cells both apical and basolateral GPI-APs associate with DRMs during their biosynthesis. However, only apical and not basolateral GPI-APs are able to oligomerize into high molecular weight complexes. Protein oligomerization begins in the medial Golgi, concomitantly with DRM association, and is dependent on protein-protein interactions. Impairment of oligomerization leads to protein missorting. We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs. Two alternative apical sorting models are presented.

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Cholesterol depletion affects HMW complex formation in the Golgi. Cells expressing GFP-GPI (A and B) or PLAP (C) were treated or not (control) with mevinolin (mev) and βCD to deplete the cells of cholesterol. Control and cholesterol-depleted cells (+ mev/βCD) expressing GFP-GPI were subjected to a temperature block in the TGN, fixed, and stained with an antibody against furin followed by a TRITC-conjugated secondary antibody. xz and xy images acquired with a confocal microscope show colocalization of GFP-GPI with the TGN marker in both control and cholesterol depleted cells (A). Control and cholesterol-depleted cells (+ mev/βCD) expressing GFP-GPI were lysed and run through a nonlinear 5–30% sucrose velocity gradient as in Fig. 3 B (B). Control and cholesterol depleted cells (+ mev/βCD) expressing PLAP were pulsed for 10 min with [35S]met, chased for 40 min and purified on velocity gradients. An aliquot of lysate was immunoprecipitated and treated with Endo H as in Fig. 5 (C).
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fig6: Cholesterol depletion affects HMW complex formation in the Golgi. Cells expressing GFP-GPI (A and B) or PLAP (C) were treated or not (control) with mevinolin (mev) and βCD to deplete the cells of cholesterol. Control and cholesterol-depleted cells (+ mev/βCD) expressing GFP-GPI were subjected to a temperature block in the TGN, fixed, and stained with an antibody against furin followed by a TRITC-conjugated secondary antibody. xz and xy images acquired with a confocal microscope show colocalization of GFP-GPI with the TGN marker in both control and cholesterol depleted cells (A). Control and cholesterol-depleted cells (+ mev/βCD) expressing GFP-GPI were lysed and run through a nonlinear 5–30% sucrose velocity gradient as in Fig. 3 B (B). Control and cholesterol depleted cells (+ mev/βCD) expressing PLAP were pulsed for 10 min with [35S]met, chased for 40 min and purified on velocity gradients. An aliquot of lysate was immunoprecipitated and treated with Endo H as in Fig. 5 (C).

Mentions: The concomitance of oligomerization and DRM association during passage of the protein through the late Golgi prompted us to evaluate whether the two events were linked by a cause–effect relation. To understand whether association to rafts was necessary for oligomer formation we impaired DRM association by depleting the cells of cholesterol (Keller and Simons, 1998; Lipardi et al., 2000) and analyzed oligomer formation in these conditions. In each experiment we obtained a depletion of 50–55% of the intracellular cholesterol using a combined treatment with mevinolin and methyl-β-cyclodextrin (βCD; see Materials and methods). As already shown for other GPI-APs, cholesterol depletion impairs both raft association and apical sorting of PLAP and GFP-GPI (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200407094/DC1). However, as described previously (Lee et al., 2002) it was difficult to see any effect on oligomeric complex formation at steady state because the proteins were already complexed in HMW complexes at the time of the treatment (unpublished data). Therefore, we analyzed the effect of cholesterol depletion on the oligomeric state of the protein in the Golgi apparatus, where oligomerization occurs and should be maximal (Fig. 5). To accumulate proteins in the TGN, control and cholesterol depleted cells expressing GFP-GPI were subjected to a temperature block at 19.5°C in the presence of cycloheximide. By double immunofluorescence with furin convertase (Liu et al., 1997) we found that GFP-GPI is highly enriched in the TGN, as shown previously (Polishchuk et al., 2004), in both control and cholesterol depleted cells (Fig. 6 A). We then subjected the cell lysates to velocity gradients and found that the ratio between the monomeric and oligomeric forms changed dramatically in cholesterol-depleted cells compared with control cells (Fig. 6 B). We observed a sevenfold decrease of the oligomeric form and a consequent increase of the monomeric form in cholesterol-depleted cells (Fig. 6 B). Because the TGN block was not tight enough for PLAP we repeated the pulse-chase and velocity gradient experiments shown in Fig. 5, but after cholesterol depletion (Fig. 6 C). As shown by the 40-min chase time point in Fig. 6 C, cholesterol depletion also affects PLAP oligomerization during its passage through the late Golgi apparatus. This suggests that rafts constitute a favorable environment for HMW complex formation of apical GPI-APs and that this event occurs during the passage of the protein through the late Golgi. Both temperature block and cholesterol depletion did not have any effect on the oligomerization of basolateral GPI-APs in that they did not oligomerize in any of these conditions (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200407094/DC1).


Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins.

Paladino S, Sarnataro D, Pillich R, Tivodar S, Nitsch L, Zurzolo C - J. Cell Biol. (2004)

Cholesterol depletion affects HMW complex formation in the Golgi. Cells expressing GFP-GPI (A and B) or PLAP (C) were treated or not (control) with mevinolin (mev) and βCD to deplete the cells of cholesterol. Control and cholesterol-depleted cells (+ mev/βCD) expressing GFP-GPI were subjected to a temperature block in the TGN, fixed, and stained with an antibody against furin followed by a TRITC-conjugated secondary antibody. xz and xy images acquired with a confocal microscope show colocalization of GFP-GPI with the TGN marker in both control and cholesterol depleted cells (A). Control and cholesterol-depleted cells (+ mev/βCD) expressing GFP-GPI were lysed and run through a nonlinear 5–30% sucrose velocity gradient as in Fig. 3 B (B). Control and cholesterol depleted cells (+ mev/βCD) expressing PLAP were pulsed for 10 min with [35S]met, chased for 40 min and purified on velocity gradients. An aliquot of lysate was immunoprecipitated and treated with Endo H as in Fig. 5 (C).
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Related In: Results  -  Collection

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fig6: Cholesterol depletion affects HMW complex formation in the Golgi. Cells expressing GFP-GPI (A and B) or PLAP (C) were treated or not (control) with mevinolin (mev) and βCD to deplete the cells of cholesterol. Control and cholesterol-depleted cells (+ mev/βCD) expressing GFP-GPI were subjected to a temperature block in the TGN, fixed, and stained with an antibody against furin followed by a TRITC-conjugated secondary antibody. xz and xy images acquired with a confocal microscope show colocalization of GFP-GPI with the TGN marker in both control and cholesterol depleted cells (A). Control and cholesterol-depleted cells (+ mev/βCD) expressing GFP-GPI were lysed and run through a nonlinear 5–30% sucrose velocity gradient as in Fig. 3 B (B). Control and cholesterol depleted cells (+ mev/βCD) expressing PLAP were pulsed for 10 min with [35S]met, chased for 40 min and purified on velocity gradients. An aliquot of lysate was immunoprecipitated and treated with Endo H as in Fig. 5 (C).
Mentions: The concomitance of oligomerization and DRM association during passage of the protein through the late Golgi prompted us to evaluate whether the two events were linked by a cause–effect relation. To understand whether association to rafts was necessary for oligomer formation we impaired DRM association by depleting the cells of cholesterol (Keller and Simons, 1998; Lipardi et al., 2000) and analyzed oligomer formation in these conditions. In each experiment we obtained a depletion of 50–55% of the intracellular cholesterol using a combined treatment with mevinolin and methyl-β-cyclodextrin (βCD; see Materials and methods). As already shown for other GPI-APs, cholesterol depletion impairs both raft association and apical sorting of PLAP and GFP-GPI (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200407094/DC1). However, as described previously (Lee et al., 2002) it was difficult to see any effect on oligomeric complex formation at steady state because the proteins were already complexed in HMW complexes at the time of the treatment (unpublished data). Therefore, we analyzed the effect of cholesterol depletion on the oligomeric state of the protein in the Golgi apparatus, where oligomerization occurs and should be maximal (Fig. 5). To accumulate proteins in the TGN, control and cholesterol depleted cells expressing GFP-GPI were subjected to a temperature block at 19.5°C in the presence of cycloheximide. By double immunofluorescence with furin convertase (Liu et al., 1997) we found that GFP-GPI is highly enriched in the TGN, as shown previously (Polishchuk et al., 2004), in both control and cholesterol depleted cells (Fig. 6 A). We then subjected the cell lysates to velocity gradients and found that the ratio between the monomeric and oligomeric forms changed dramatically in cholesterol-depleted cells compared with control cells (Fig. 6 B). We observed a sevenfold decrease of the oligomeric form and a consequent increase of the monomeric form in cholesterol-depleted cells (Fig. 6 B). Because the TGN block was not tight enough for PLAP we repeated the pulse-chase and velocity gradient experiments shown in Fig. 5, but after cholesterol depletion (Fig. 6 C). As shown by the 40-min chase time point in Fig. 6 C, cholesterol depletion also affects PLAP oligomerization during its passage through the late Golgi apparatus. This suggests that rafts constitute a favorable environment for HMW complex formation of apical GPI-APs and that this event occurs during the passage of the protein through the late Golgi. Both temperature block and cholesterol depletion did not have any effect on the oligomerization of basolateral GPI-APs in that they did not oligomerize in any of these conditions (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200407094/DC1).

Bottom Line: Impairment of oligomerization leads to protein missorting.We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs.Two alternative apical sorting models are presented.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia e Patologia Cellulare e Molecolare, Centro di Endocrinologia ed Oncologia Sperimentale, CNR, Università degli Studi di Napoli Federico II, Italy.

ABSTRACT
An essential but insufficient step for apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in epithelial cells is their association with detergent-resistant microdomains (DRMs) or rafts. In this paper, we show that in MDCK cells both apical and basolateral GPI-APs associate with DRMs during their biosynthesis. However, only apical and not basolateral GPI-APs are able to oligomerize into high molecular weight complexes. Protein oligomerization begins in the medial Golgi, concomitantly with DRM association, and is dependent on protein-protein interactions. Impairment of oligomerization leads to protein missorting. We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs. Two alternative apical sorting models are presented.

Show MeSH
Related in: MedlinePlus