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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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Only apical GPI-APs can be cross-linked at the cell surface. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF grown on filters were incubated with BS3 (0.5 mM). After lysis, proteins were TCA precipitated, run on SDS-PAGE (in a 6–12% gradient gel for GFP-GPI, PrP, or GH-DAF or 8% gel for PLAP) in reducing conditions and revealed with specific antibodies. The molecular weight of the monomeric forms (*) of each protein is indicated, together with the position of a 180-kD marker. ** and *** indicate, respectively, the expected molecular weight of the dimeric and trimeric forms of each protein.
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fig4: Only apical GPI-APs can be cross-linked at the cell surface. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF grown on filters were incubated with BS3 (0.5 mM). After lysis, proteins were TCA precipitated, run on SDS-PAGE (in a 6–12% gradient gel for GFP-GPI, PrP, or GH-DAF or 8% gel for PLAP) in reducing conditions and revealed with specific antibodies. The molecular weight of the monomeric forms (*) of each protein is indicated, together with the position of a 180-kD marker. ** and *** indicate, respectively, the expected molecular weight of the dimeric and trimeric forms of each protein.

Mentions: To rule out the possibility that these HMW complexes were formed as a consequence of detergent addition to the cells we used a different approach in native conditions. Therefore, we added a chemical cross-linking agent, bis(sulfosuccinimidyl)suberate (BS3), that is able to cross-link molecules that are in very close proximity (arm length, 11.4 Å; Friedrichson and Kurzchalia, 1998) either to the apical or the basolateral surface of filters grown cells. As expected, we did not find HMW complexes in the absence of cross-linking (Fig. 4). However, when cells expressing GFP-GPI were chemically cross-linked at 4°C from the apical surface, a smear between ∼80 and ∼300 kD was detected on the gel (Fig. 4). Similarly, we detected a band corresponding to a relative molecular mass of 120 kD (dimer), a band of 180 kD (trimer), and a smear at higher molecular weights after apical cross-linking of PLAP-expressing cells (Fig. 4). In contrast, neither PrP nor GH-DAF were found in cross-linked complexes when BS3 was added to the basolateral surface (Fig. 4). Because BS3 is membrane impermeable, our data indicate that apical GPI-APs are in cross-linkable complexes at the cell surface, whereas basolateral ones are not.


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)

Only apical GPI-APs can be cross-linked at the cell surface. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF grown on filters were incubated with BS3 (0.5 mM). After lysis, proteins were TCA precipitated, run on SDS-PAGE (in a 6–12% gradient gel for GFP-GPI, PrP, or GH-DAF or 8% gel for PLAP) in reducing conditions and revealed with specific antibodies. The molecular weight of the monomeric forms (*) of each protein is indicated, together with the position of a 180-kD marker. ** and *** indicate, respectively, the expected molecular weight of the dimeric and trimeric forms of each protein.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172584&req=5

fig4: Only apical GPI-APs can be cross-linked at the cell surface. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF grown on filters were incubated with BS3 (0.5 mM). After lysis, proteins were TCA precipitated, run on SDS-PAGE (in a 6–12% gradient gel for GFP-GPI, PrP, or GH-DAF or 8% gel for PLAP) in reducing conditions and revealed with specific antibodies. The molecular weight of the monomeric forms (*) of each protein is indicated, together with the position of a 180-kD marker. ** and *** indicate, respectively, the expected molecular weight of the dimeric and trimeric forms of each protein.
Mentions: To rule out the possibility that these HMW complexes were formed as a consequence of detergent addition to the cells we used a different approach in native conditions. Therefore, we added a chemical cross-linking agent, bis(sulfosuccinimidyl)suberate (BS3), that is able to cross-link molecules that are in very close proximity (arm length, 11.4 Å; Friedrichson and Kurzchalia, 1998) either to the apical or the basolateral surface of filters grown cells. As expected, we did not find HMW complexes in the absence of cross-linking (Fig. 4). However, when cells expressing GFP-GPI were chemically cross-linked at 4°C from the apical surface, a smear between ∼80 and ∼300 kD was detected on the gel (Fig. 4). Similarly, we detected a band corresponding to a relative molecular mass of 120 kD (dimer), a band of 180 kD (trimer), and a smear at higher molecular weights after apical cross-linking of PLAP-expressing cells (Fig. 4). In contrast, neither PrP nor GH-DAF were found in cross-linked complexes when BS3 was added to the basolateral surface (Fig. 4). Because BS3 is membrane impermeable, our data indicate that apical GPI-APs are in cross-linkable complexes at the cell surface, whereas basolateral ones are not.

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