Limits...
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

Related in: MedlinePlus

Both apical and basolateral GPI-APs associate with DRM. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were lysed in TNE/TX-100 buffer at 4°C and separated by centrifugation into soluble and insoluble fractions (A) or run through 5–40% sucrose gradients (B). Fractions of 1 ml were collected from top (fraction 1) to bottom (fraction 12) after centrifugation to equilibrium (B). After TCA precipitation samples were run on SDS-PAGE and detected by specific antibodies. An aliquot of each fraction was spotted on the nitrocellulose membrane and GM1 was revealed using cholera toxin conjugated to HRP. The histograms in A show the percent of soluble or insoluble protein from three different experiments (standard error bars are indicated). Note that the slower mobility band (43 kD) detected for GFP-GPI is specific for GFP because it is not present in untransfected cells (unpublished data) and it was described previously as a partially denaturated GFP dimer (Inouye and Tsuji, 1994). PrP migrates as four major bands: the mature fully glycosylated form corresponding to the molecular mass of 31 kD, the immature glycosylated forms (diglycosylated and monoglycosylated) and the unglycosylated form (Sarnataro et al., 2002, 2004).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172584&req=5

fig2: Both apical and basolateral GPI-APs associate with DRM. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were lysed in TNE/TX-100 buffer at 4°C and separated by centrifugation into soluble and insoluble fractions (A) or run through 5–40% sucrose gradients (B). Fractions of 1 ml were collected from top (fraction 1) to bottom (fraction 12) after centrifugation to equilibrium (B). After TCA precipitation samples were run on SDS-PAGE and detected by specific antibodies. An aliquot of each fraction was spotted on the nitrocellulose membrane and GM1 was revealed using cholera toxin conjugated to HRP. The histograms in A show the percent of soluble or insoluble protein from three different experiments (standard error bars are indicated). Note that the slower mobility band (43 kD) detected for GFP-GPI is specific for GFP because it is not present in untransfected cells (unpublished data) and it was described previously as a partially denaturated GFP dimer (Inouye and Tsuji, 1994). PrP migrates as four major bands: the mature fully glycosylated form corresponding to the molecular mass of 31 kD, the immature glycosylated forms (diglycosylated and monoglycosylated) and the unglycosylated form (Sarnataro et al., 2002, 2004).

Mentions: It has been postulated that in MDCK cells GPI-APs are sorted to the apical surface through their incorporation into lipid microdomains (rafts) in the Golgi complex (Simons and Ikonen, 1997). Rafts can be isolated from whole cells as membranes resistant to extraction in cold nonionic detergents (detergent resistant membrane [DRM]) such as TX-100 (Edidin, 2003; Helms and Zurzolo, 2004; Simons and Vaz, 2004). To understand the role of DRM association for apical sorting of GPI-APs we extracted the different MDCK clones expressing GFP-GPI, PLAP, PrP, and GH-DAF in cold TX-100, as described previously (Brown and Rose, 1992; Zurzolo et al., 1994). We found that both apical and basolateral GPI-APs were insoluble to TX-100 extraction (respectively, ∼75–80% for the apical proteins and ∼60–70% for the basolateral ones; Fig. 2 A). Because TX-100 insolubility can also result from events other than DRM association (Low and Saltiel, 1988; Brown and Rose, 1992), we purified TX-100 insoluble microdomains by centrifugation to equilibrium on sucrose density gradients, that allows the segregation of lipid-rich components from the bulk of TX-100 insoluble material (Brown and Rose, 1992). Consistently with the TX-100 extraction results (Fig. 2 A) we found that all four GPI-APs floated to the DRM-GM1–enriched fractions (4–7) of the gradients (Fig. 2 B). Therefore, these experiments clearly confirmed that association to DRMs is not sufficient to dictate apical sorting (Benting et al., 1999b; Lipardi et al., 2000; Sarnataro et al., 2002).


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)

Both apical and basolateral GPI-APs associate with DRM. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were lysed in TNE/TX-100 buffer at 4°C and separated by centrifugation into soluble and insoluble fractions (A) or run through 5–40% sucrose gradients (B). Fractions of 1 ml were collected from top (fraction 1) to bottom (fraction 12) after centrifugation to equilibrium (B). After TCA precipitation samples were run on SDS-PAGE and detected by specific antibodies. An aliquot of each fraction was spotted on the nitrocellulose membrane and GM1 was revealed using cholera toxin conjugated to HRP. The histograms in A show the percent of soluble or insoluble protein from three different experiments (standard error bars are indicated). Note that the slower mobility band (43 kD) detected for GFP-GPI is specific for GFP because it is not present in untransfected cells (unpublished data) and it was described previously as a partially denaturated GFP dimer (Inouye and Tsuji, 1994). PrP migrates as four major bands: the mature fully glycosylated form corresponding to the molecular mass of 31 kD, the immature glycosylated forms (diglycosylated and monoglycosylated) and the unglycosylated form (Sarnataro et al., 2002, 2004).
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Both apical and basolateral GPI-APs associate with DRM. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were lysed in TNE/TX-100 buffer at 4°C and separated by centrifugation into soluble and insoluble fractions (A) or run through 5–40% sucrose gradients (B). Fractions of 1 ml were collected from top (fraction 1) to bottom (fraction 12) after centrifugation to equilibrium (B). After TCA precipitation samples were run on SDS-PAGE and detected by specific antibodies. An aliquot of each fraction was spotted on the nitrocellulose membrane and GM1 was revealed using cholera toxin conjugated to HRP. The histograms in A show the percent of soluble or insoluble protein from three different experiments (standard error bars are indicated). Note that the slower mobility band (43 kD) detected for GFP-GPI is specific for GFP because it is not present in untransfected cells (unpublished data) and it was described previously as a partially denaturated GFP dimer (Inouye and Tsuji, 1994). PrP migrates as four major bands: the mature fully glycosylated form corresponding to the molecular mass of 31 kD, the immature glycosylated forms (diglycosylated and monoglycosylated) and the unglycosylated form (Sarnataro et al., 2002, 2004).
Mentions: It has been postulated that in MDCK cells GPI-APs are sorted to the apical surface through their incorporation into lipid microdomains (rafts) in the Golgi complex (Simons and Ikonen, 1997). Rafts can be isolated from whole cells as membranes resistant to extraction in cold nonionic detergents (detergent resistant membrane [DRM]) such as TX-100 (Edidin, 2003; Helms and Zurzolo, 2004; Simons and Vaz, 2004). To understand the role of DRM association for apical sorting of GPI-APs we extracted the different MDCK clones expressing GFP-GPI, PLAP, PrP, and GH-DAF in cold TX-100, as described previously (Brown and Rose, 1992; Zurzolo et al., 1994). We found that both apical and basolateral GPI-APs were insoluble to TX-100 extraction (respectively, ∼75–80% for the apical proteins and ∼60–70% for the basolateral ones; Fig. 2 A). Because TX-100 insolubility can also result from events other than DRM association (Low and Saltiel, 1988; Brown and Rose, 1992), we purified TX-100 insoluble microdomains by centrifugation to equilibrium on sucrose density gradients, that allows the segregation of lipid-rich components from the bulk of TX-100 insoluble material (Brown and Rose, 1992). Consistently with the TX-100 extraction results (Fig. 2 A) we found that all four GPI-APs floated to the DRM-GM1–enriched fractions (4–7) of the gradients (Fig. 2 B). Therefore, these experiments clearly confirmed that association to DRMs is not sufficient to dictate apical sorting (Benting et al., 1999b; Lipardi et al., 2000; Sarnataro et al., 2002).

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