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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.

Show MeSH
GPI-APs are apically and basolaterally sorted. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were grown to confluence on filters. Cells were fixed and in the case of PLAP, PrP, and GH-DAF stained with specific antibodies followed by a TRITC-conjugated secondary antibody in nonpermeabilized conditions. Serial confocal sections were collected from the top to the bottom of cell monolayers (A). Cells were labeled with LC-biotin respectively added to the apical or the basolateral surface. After immunoprecipitation with specific antibodies samples were run on SDS-PAGE and revealed using HRP-streptavidin (B). The histograms show percentages of apical or basolateral protein expressed as the average of three different experiments. Standard error bars are indicated.
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fig1: GPI-APs are apically and basolaterally sorted. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were grown to confluence on filters. Cells were fixed and in the case of PLAP, PrP, and GH-DAF stained with specific antibodies followed by a TRITC-conjugated secondary antibody in nonpermeabilized conditions. Serial confocal sections were collected from the top to the bottom of cell monolayers (A). Cells were labeled with LC-biotin respectively added to the apical or the basolateral surface. After immunoprecipitation with specific antibodies samples were run on SDS-PAGE and revealed using HRP-streptavidin (B). The histograms show percentages of apical or basolateral protein expressed as the average of three different experiments. Standard error bars are indicated.

Mentions: By confocal microscopy (Fig. 1 A) and surface biotinylation (Fig. 1 B) we confirmed that GFP-GPI and PLAP were predominantly enriched on the apical surface, whereas PrP and GH-DAF were mainly localized on the basolateral membrane (Arreaza and Brown, 1995; Benting et al., 1999b; Sarnataro et al., 2002; Polishchuk et al., 2004).


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)

GPI-APs are apically and basolaterally sorted. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were grown to confluence on filters. Cells were fixed and in the case of PLAP, PrP, and GH-DAF stained with specific antibodies followed by a TRITC-conjugated secondary antibody in nonpermeabilized conditions. Serial confocal sections were collected from the top to the bottom of cell monolayers (A). Cells were labeled with LC-biotin respectively added to the apical or the basolateral surface. After immunoprecipitation with specific antibodies samples were run on SDS-PAGE and revealed using HRP-streptavidin (B). The histograms show percentages of apical or basolateral protein expressed as the average of three different experiments. Standard error bars are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: GPI-APs are apically and basolaterally sorted. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were grown to confluence on filters. Cells were fixed and in the case of PLAP, PrP, and GH-DAF stained with specific antibodies followed by a TRITC-conjugated secondary antibody in nonpermeabilized conditions. Serial confocal sections were collected from the top to the bottom of cell monolayers (A). Cells were labeled with LC-biotin respectively added to the apical or the basolateral surface. After immunoprecipitation with specific antibodies samples were run on SDS-PAGE and revealed using HRP-streptavidin (B). The histograms show percentages of apical or basolateral protein expressed as the average of three different experiments. Standard error bars are indicated.
Mentions: By confocal microscopy (Fig. 1 A) and surface biotinylation (Fig. 1 B) we confirmed that GFP-GPI and PLAP were predominantly enriched on the apical surface, whereas PrP and GH-DAF were mainly localized on the basolateral membrane (Arreaza and Brown, 1995; Benting et al., 1999b; Sarnataro et al., 2002; Polishchuk et al., 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.

Show MeSH