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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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Oligomerization impairment leads GFP-GPI missorting. MDCK cells expressing GFP-GPI or the double cysteine GFP-GPI mutant (S49/71) were lysed in TNE/TX-100 buffer and samples were run on SDS-PAGE in nonreducing conditions. Although GFP-GPI migrates as monomeric and HMW forms, the S49/71 mutant runs exclusively as a monomer (A). White lines indicate that intervening lanes have been spliced out. (B) MDCK cells expressing the S49/71 mutant of GFP-GPI were purified on velocity gradients at the steady state (top) and after temperature block in the TGN (bottom). After TCA precipitation the protein was revealed in the different fractions by Western blotting using an anti-GFP antibody. Also in this assay the mutant migrates almost exclusively as a monomer. MDCK cells expressing S49/71 mutant of GFP-GPI were lysed in TNE/TX-100 buffer at 4°C and subjected to flotation by centrifugation to equilibrium on sucrose gradients as described before. The collected fractions were TCA precipitated and proteins were revealed using an antibody against GFP (C). (D) MDCK cells expressing wild-type and double cysteine mutant of GFP-GPI were grown on filters in polarized conditions for 4 d and stained using an antibody against c-Myc in nonpermeabilizing conditions followed by a TRITC-conjugated secondary antibody. Images were collected with a confocal microscope. xy images shown are taken at the top or at the bottom of the cells (left). xz images are also shown in top left panels. Filter grown cells were biotinylated as in Fig. 1 B. The histograms show percentages of apical or basolateral protein expressed as the average of three different experiments. Standard error bars are indicated (right).
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fig7: Oligomerization impairment leads GFP-GPI missorting. MDCK cells expressing GFP-GPI or the double cysteine GFP-GPI mutant (S49/71) were lysed in TNE/TX-100 buffer and samples were run on SDS-PAGE in nonreducing conditions. Although GFP-GPI migrates as monomeric and HMW forms, the S49/71 mutant runs exclusively as a monomer (A). White lines indicate that intervening lanes have been spliced out. (B) MDCK cells expressing the S49/71 mutant of GFP-GPI were purified on velocity gradients at the steady state (top) and after temperature block in the TGN (bottom). After TCA precipitation the protein was revealed in the different fractions by Western blotting using an anti-GFP antibody. Also in this assay the mutant migrates almost exclusively as a monomer. MDCK cells expressing S49/71 mutant of GFP-GPI were lysed in TNE/TX-100 buffer at 4°C and subjected to flotation by centrifugation to equilibrium on sucrose gradients as described before. The collected fractions were TCA precipitated and proteins were revealed using an antibody against GFP (C). (D) MDCK cells expressing wild-type and double cysteine mutant of GFP-GPI were grown on filters in polarized conditions for 4 d and stained using an antibody against c-Myc in nonpermeabilizing conditions followed by a TRITC-conjugated secondary antibody. Images were collected with a confocal microscope. xy images shown are taken at the top or at the bottom of the cells (left). xz images are also shown in top left panels. Filter grown cells were biotinylated as in Fig. 1 B. The histograms show percentages of apical or basolateral protein expressed as the average of three different experiments. Standard error bars are indicated (right).

Mentions: To understand whether GPI-APs clustering in HMW complexes has a key role in their apical sorting, we decided to impair oligomerization and study its effect on apical sorting. It was recently described that GFP oligomerizes in the secretory pathway and that GFP oligomers depend on disulphide bonds (Jain et al., 2001). Because two specific cysteines, cys 49 and cys 71, are involved in GFP oligomerization (Jain et al., 2001), we mutated them in the GFP-GPI construct by site-directed mutagenesis. In contrast to the wild-type, the double cys GFP-GPI mutant (S49/71) ran exclusively as a monomer in both nonreducing gels (Fig. 7 A) and on velocity gradients (Fig. 7 B) both at steady state (top) and after the block in the TGN (bottom) indicating that it was not able to oligomerize. However, similar to the wild-type protein, ∼70% of the S49/71 mutant was TX-100 insoluble (unpublished data) and ∼60% floated to the lighter fractions on sucrose density gradients (Fig. 7 C), although there was a shift toward the bottom of the gradient of the mutated protein compared with the wild-type (compare Fig. 2 B with Fig. 7 C). Thus the two point mutations do not affect GFP-GPI association with DRMs, suggesting that they did not dramatically alter the structure of the protein (because it was immunoprecipiated with similar affinity as the wild-type protein (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200407094/DC1) nor impair its transport to the plasma membrane, as shown below (Fig. 7 D).


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)

Oligomerization impairment leads GFP-GPI missorting. MDCK cells expressing GFP-GPI or the double cysteine GFP-GPI mutant (S49/71) were lysed in TNE/TX-100 buffer and samples were run on SDS-PAGE in nonreducing conditions. Although GFP-GPI migrates as monomeric and HMW forms, the S49/71 mutant runs exclusively as a monomer (A). White lines indicate that intervening lanes have been spliced out. (B) MDCK cells expressing the S49/71 mutant of GFP-GPI were purified on velocity gradients at the steady state (top) and after temperature block in the TGN (bottom). After TCA precipitation the protein was revealed in the different fractions by Western blotting using an anti-GFP antibody. Also in this assay the mutant migrates almost exclusively as a monomer. MDCK cells expressing S49/71 mutant of GFP-GPI were lysed in TNE/TX-100 buffer at 4°C and subjected to flotation by centrifugation to equilibrium on sucrose gradients as described before. The collected fractions were TCA precipitated and proteins were revealed using an antibody against GFP (C). (D) MDCK cells expressing wild-type and double cysteine mutant of GFP-GPI were grown on filters in polarized conditions for 4 d and stained using an antibody against c-Myc in nonpermeabilizing conditions followed by a TRITC-conjugated secondary antibody. Images were collected with a confocal microscope. xy images shown are taken at the top or at the bottom of the cells (left). xz images are also shown in top left panels. Filter grown cells were biotinylated as in Fig. 1 B. The histograms show percentages of apical or basolateral protein expressed as the average of three different experiments. Standard error bars are indicated (right).
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Related In: Results  -  Collection

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fig7: Oligomerization impairment leads GFP-GPI missorting. MDCK cells expressing GFP-GPI or the double cysteine GFP-GPI mutant (S49/71) were lysed in TNE/TX-100 buffer and samples were run on SDS-PAGE in nonreducing conditions. Although GFP-GPI migrates as monomeric and HMW forms, the S49/71 mutant runs exclusively as a monomer (A). White lines indicate that intervening lanes have been spliced out. (B) MDCK cells expressing the S49/71 mutant of GFP-GPI were purified on velocity gradients at the steady state (top) and after temperature block in the TGN (bottom). After TCA precipitation the protein was revealed in the different fractions by Western blotting using an anti-GFP antibody. Also in this assay the mutant migrates almost exclusively as a monomer. MDCK cells expressing S49/71 mutant of GFP-GPI were lysed in TNE/TX-100 buffer at 4°C and subjected to flotation by centrifugation to equilibrium on sucrose gradients as described before. The collected fractions were TCA precipitated and proteins were revealed using an antibody against GFP (C). (D) MDCK cells expressing wild-type and double cysteine mutant of GFP-GPI were grown on filters in polarized conditions for 4 d and stained using an antibody against c-Myc in nonpermeabilizing conditions followed by a TRITC-conjugated secondary antibody. Images were collected with a confocal microscope. xy images shown are taken at the top or at the bottom of the cells (left). xz images are also shown in top left panels. Filter grown cells were biotinylated as in Fig. 1 B. The histograms show percentages of apical or basolateral protein expressed as the average of three different experiments. Standard error bars are indicated (right).
Mentions: To understand whether GPI-APs clustering in HMW complexes has a key role in their apical sorting, we decided to impair oligomerization and study its effect on apical sorting. It was recently described that GFP oligomerizes in the secretory pathway and that GFP oligomers depend on disulphide bonds (Jain et al., 2001). Because two specific cysteines, cys 49 and cys 71, are involved in GFP oligomerization (Jain et al., 2001), we mutated them in the GFP-GPI construct by site-directed mutagenesis. In contrast to the wild-type, the double cys GFP-GPI mutant (S49/71) ran exclusively as a monomer in both nonreducing gels (Fig. 7 A) and on velocity gradients (Fig. 7 B) both at steady state (top) and after the block in the TGN (bottom) indicating that it was not able to oligomerize. However, similar to the wild-type protein, ∼70% of the S49/71 mutant was TX-100 insoluble (unpublished data) and ∼60% floated to the lighter fractions on sucrose density gradients (Fig. 7 C), although there was a shift toward the bottom of the gradient of the mutated protein compared with the wild-type (compare Fig. 2 B with Fig. 7 C). Thus the two point mutations do not affect GFP-GPI association with DRMs, suggesting that they did not dramatically alter the structure of the protein (because it was immunoprecipiated with similar affinity as the wild-type protein (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200407094/DC1) nor impair its transport to the plasma membrane, as shown below (Fig. 7 D).

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