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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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HMW complex formation occurs concomitantly with DRM association of the protein. MDCK cells expressing PLAP or PrP grown on plastic dishes were pulsed for 10 min with [35S]cys and -met and chased for the indicated times. At the end of each chase time the cells were lysed and purified on velocity gradients (left) or on sucrose density gradients (right). For each chase time an aliquot of lysate was immunoprecipitated and treated with Endo H. H, mature highly glycosylated; D, diglycosylated; M, monoglycosylated; U, unglycosylated PrP isoforms (Sarnataro et al., 2002, 2004). Note that up to 10 min of chase PLAP was not found in HMW complex, by 20 and 40 min of chase ∼10% and ∼25–30%, respectively, of PLAP was in HMW complex. At 80 min PLAP was still in these complexes (∼15–20%). In contrast, all PrP was purified exclusively as monomer form at all chase times. White lines indicate that intervening lanes have been spliced out.
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fig5: HMW complex formation occurs concomitantly with DRM association of the protein. MDCK cells expressing PLAP or PrP grown on plastic dishes were pulsed for 10 min with [35S]cys and -met and chased for the indicated times. At the end of each chase time the cells were lysed and purified on velocity gradients (left) or on sucrose density gradients (right). For each chase time an aliquot of lysate was immunoprecipitated and treated with Endo H. H, mature highly glycosylated; D, diglycosylated; M, monoglycosylated; U, unglycosylated PrP isoforms (Sarnataro et al., 2002, 2004). Note that up to 10 min of chase PLAP was not found in HMW complex, by 20 and 40 min of chase ∼10% and ∼25–30%, respectively, of PLAP was in HMW complex. At 80 min PLAP was still in these complexes (∼15–20%). In contrast, all PrP was purified exclusively as monomer form at all chase times. White lines indicate that intervening lanes have been spliced out.

Mentions: To study when and where apical GPI-APs were oligomerizing during their life span and to understand whether this event had any role in apical sorting, we analyzed the kinetics of GPI-APs oligomerization by pulse-chase experiments (Fig. 5, left). Although we obtained overlapping data for GFP-GPI and PLAP, we only show the oligomer formation of PLAP (Fig. 5) because it is glycosylated and therefore it was possible to monitor its passage through the Golgi apparatus by acquisition of resistance to endoglycosidase H (Endo H) digestion (Kornfeld and Kornfeld, 1985). After a brief pulse of 10 min with [35S]met/cys, cells were chased for the indicated times, lysed in SDS/TX-100 containing buffer, and run on velocity gradients (Fig. 5, left). PLAP began to form HMW complexes after 20 min of chase when a portion of the protein had acquired Endo H resistance (therefore after the medial Golgi). After 40 min of chase, when almost all PLAP was Endo H resistant, ∼30% of the protein was found in HMW complexes (Fig. 5, top left). Interestingly, PLAP was recovered in HMW complexes (although in lower amounts, ∼20%) also after 80 min of chase, i.e., when the majority of the protein had already reached the plasma membrane, as shown by our targeting assays (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200407094/DC1). Our results are in agreement with previous data (Friedrichson and Kurzchalia, 1998; Harder et al., 1998; Varma and Mayor, 1998) showing that GPI-APs are in clusters at the cell surface, in addition they demonstrate that apical GPI-APs cluster during their passage through the Golgi where sorting is supposed to occur (Wandinger-Ness et al., 1990; Rodriguez-Boulan and Powell, 1992; Mostov et al., 2000; Keller et al., 2001).


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)

HMW complex formation occurs concomitantly with DRM association of the protein. MDCK cells expressing PLAP or PrP grown on plastic dishes were pulsed for 10 min with [35S]cys and -met and chased for the indicated times. At the end of each chase time the cells were lysed and purified on velocity gradients (left) or on sucrose density gradients (right). For each chase time an aliquot of lysate was immunoprecipitated and treated with Endo H. H, mature highly glycosylated; D, diglycosylated; M, monoglycosylated; U, unglycosylated PrP isoforms (Sarnataro et al., 2002, 2004). Note that up to 10 min of chase PLAP was not found in HMW complex, by 20 and 40 min of chase ∼10% and ∼25–30%, respectively, of PLAP was in HMW complex. At 80 min PLAP was still in these complexes (∼15–20%). In contrast, all PrP was purified exclusively as monomer form at all chase times. White lines indicate that intervening lanes have been spliced out.
© Copyright Policy
Related In: Results  -  Collection

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fig5: HMW complex formation occurs concomitantly with DRM association of the protein. MDCK cells expressing PLAP or PrP grown on plastic dishes were pulsed for 10 min with [35S]cys and -met and chased for the indicated times. At the end of each chase time the cells were lysed and purified on velocity gradients (left) or on sucrose density gradients (right). For each chase time an aliquot of lysate was immunoprecipitated and treated with Endo H. H, mature highly glycosylated; D, diglycosylated; M, monoglycosylated; U, unglycosylated PrP isoforms (Sarnataro et al., 2002, 2004). Note that up to 10 min of chase PLAP was not found in HMW complex, by 20 and 40 min of chase ∼10% and ∼25–30%, respectively, of PLAP was in HMW complex. At 80 min PLAP was still in these complexes (∼15–20%). In contrast, all PrP was purified exclusively as monomer form at all chase times. White lines indicate that intervening lanes have been spliced out.
Mentions: To study when and where apical GPI-APs were oligomerizing during their life span and to understand whether this event had any role in apical sorting, we analyzed the kinetics of GPI-APs oligomerization by pulse-chase experiments (Fig. 5, left). Although we obtained overlapping data for GFP-GPI and PLAP, we only show the oligomer formation of PLAP (Fig. 5) because it is glycosylated and therefore it was possible to monitor its passage through the Golgi apparatus by acquisition of resistance to endoglycosidase H (Endo H) digestion (Kornfeld and Kornfeld, 1985). After a brief pulse of 10 min with [35S]met/cys, cells were chased for the indicated times, lysed in SDS/TX-100 containing buffer, and run on velocity gradients (Fig. 5, left). PLAP began to form HMW complexes after 20 min of chase when a portion of the protein had acquired Endo H resistance (therefore after the medial Golgi). After 40 min of chase, when almost all PLAP was Endo H resistant, ∼30% of the protein was found in HMW complexes (Fig. 5, top left). Interestingly, PLAP was recovered in HMW complexes (although in lower amounts, ∼20%) also after 80 min of chase, i.e., when the majority of the protein had already reached the plasma membrane, as shown by our targeting assays (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200407094/DC1). Our results are in agreement with previous data (Friedrichson and Kurzchalia, 1998; Harder et al., 1998; Varma and Mayor, 1998) showing that GPI-APs are in clusters at the cell surface, in addition they demonstrate that apical GPI-APs cluster during their passage through the Golgi where sorting is supposed to occur (Wandinger-Ness et al., 1990; Rodriguez-Boulan and Powell, 1992; Mostov et al., 2000; Keller et al., 2001).

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