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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 form HMW complexes. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were lysed in buffer containing 0.4% SDS and 0.2% TX-100 and run through a nonlinear 5–30% sucrose gradient. Fractions of 500 μl were collected from the top (fraction 1) to the bottom (fraction 9) of the gradients. Proteins were TCA precipitated and detected by Western blotting using specific antibodies. The molecular weight of the monomeric forms of each protein is indicated. The position on the gradients of molecular weight markers is indicated on the top of the panel. Distribution curves of the average of three different experiments (standard error bars are indicated) are shown in the right panel.
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fig3: Only apical GPI-APs form HMW complexes. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were lysed in buffer containing 0.4% SDS and 0.2% TX-100 and run through a nonlinear 5–30% sucrose gradient. Fractions of 500 μl were collected from the top (fraction 1) to the bottom (fraction 9) of the gradients. Proteins were TCA precipitated and detected by Western blotting using specific antibodies. The molecular weight of the monomeric forms of each protein is indicated. The position on the gradients of molecular weight markers is indicated on the top of the panel. Distribution curves of the average of three different experiments (standard error bars are indicated) are shown in the right panel.

Mentions: The differences in DRM association observed for apical and basolateral GPI-APs could be due to a different lipid environment surrounding the different proteins or to a different affinity for DRMs of apical and basolateral GPI-APs. In the latter case a high affinity could lead to a more stable association of the protein with lipid rafts which could promote apical sorting. Hence, it has been shown that clustering of seven or fewer GPI-APs increases their raft association and this leads to their rerouting to late endosomes instead of recycling endosomes (Fivaz et al., 2002). Because it is well known that protein multimers partition preferentially in DRMs compared with their monomeric form (Fivaz et al., 2002; Cunningham et al., 2003; Helms and Zurzolo, 2004; Simons and Vaz, 2004), we decided to analyze the oligomerization state of the different apical and basolateral GPI-APs by sedimentation on velocity gradients (where the proteins sediment according to their molecular weight) after extraction in SDS/TX-100 buffer (Scheiffele et al., 1998). Although ∼20–30% of GFP-GPI and ∼25–35% of PLAP were purified as HMW complexes containing more than a trimer, both PrP and GH-DAF were purified almost exclusively from the gradient fractions corresponding to their expected monomeric molecular weights (Fig. 3). Therefore, these experiments revealed that only apical GPI-APs are in oligomeric complexes.


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 form HMW complexes. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were lysed in buffer containing 0.4% SDS and 0.2% TX-100 and run through a nonlinear 5–30% sucrose gradient. Fractions of 500 μl were collected from the top (fraction 1) to the bottom (fraction 9) of the gradients. Proteins were TCA precipitated and detected by Western blotting using specific antibodies. The molecular weight of the monomeric forms of each protein is indicated. The position on the gradients of molecular weight markers is indicated on the top of the panel. Distribution curves of the average of three different experiments (standard error bars are indicated) are shown in the right panel.
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Related In: Results  -  Collection

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fig3: Only apical GPI-APs form HMW complexes. MDCK cells stably expressing GFP-GPI, PLAP, PrP, or GH-DAF were lysed in buffer containing 0.4% SDS and 0.2% TX-100 and run through a nonlinear 5–30% sucrose gradient. Fractions of 500 μl were collected from the top (fraction 1) to the bottom (fraction 9) of the gradients. Proteins were TCA precipitated and detected by Western blotting using specific antibodies. The molecular weight of the monomeric forms of each protein is indicated. The position on the gradients of molecular weight markers is indicated on the top of the panel. Distribution curves of the average of three different experiments (standard error bars are indicated) are shown in the right panel.
Mentions: The differences in DRM association observed for apical and basolateral GPI-APs could be due to a different lipid environment surrounding the different proteins or to a different affinity for DRMs of apical and basolateral GPI-APs. In the latter case a high affinity could lead to a more stable association of the protein with lipid rafts which could promote apical sorting. Hence, it has been shown that clustering of seven or fewer GPI-APs increases their raft association and this leads to their rerouting to late endosomes instead of recycling endosomes (Fivaz et al., 2002). Because it is well known that protein multimers partition preferentially in DRMs compared with their monomeric form (Fivaz et al., 2002; Cunningham et al., 2003; Helms and Zurzolo, 2004; Simons and Vaz, 2004), we decided to analyze the oligomerization state of the different apical and basolateral GPI-APs by sedimentation on velocity gradients (where the proteins sediment according to their molecular weight) after extraction in SDS/TX-100 buffer (Scheiffele et al., 1998). Although ∼20–30% of GFP-GPI and ∼25–35% of PLAP were purified as HMW complexes containing more than a trimer, both PrP and GH-DAF were purified almost exclusively from the gradient fractions corresponding to their expected monomeric molecular weights (Fig. 3). Therefore, these experiments revealed that only apical GPI-APs are in oligomeric complexes.

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