Detergent-insoluble GPI-anchored proteins are apically sorted in fischer rat thyroid cells, but interference with cholesterol or sphingolipids differentially affects detergent insolubility and apical sorting.
In contrast to Madin-Darby canine kidney cells, Fischer rat thyroid cells deliver the majority of endogenous glycosylphosphatidyl inositol (GPI)-anchored proteins to the basolateral surface. However, we report here that the GPI proteins Placental Alkaline Phosphatase (PLAP) and Neurotrophin Receptor-Placental Alkaline Phosphatase (NTR-PLAP) are apically localized in transfected Fischer rat thyroid cells. In agreement with the "raft hypothesis," which postulates the incorporation of GPI proteins into glycosphingolipids and cholesterol-enriched rafts, we found that both of these proteins were insoluble in Triton X-100 and floated into the lighter fractions of sucrose density gradients. However, disruption of lipid rafts by removal of cholesterol did not cause surface missorting of PLAP and NTR-PLAP, and the altered surface sorting of these proteins after Fumonisin B1 treatment did not correlate with reduced levels in Triton X-100 -insoluble fractions. Furthermore, in contrast to the GPI-anchored forms of both of these proteins, the secretory and transmembrane forms (in the absence of a basolateral cytoplasmic signal) were sorted to the apical surface without association with lipid microdomains. Together, these data demonstrate that the GPI anchor is required to mediate raft association but is not sufficient to determine apical sorting. They also suggest that signals present in the ectodomain of the proteins play a major role and that lipid rafts may facilitate the recognition of these signals in the trans-Golgi network, even though they are not required for apical sorting.
Lipardi
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Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche-Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli "Federico II," 80131 Napoli, Italy.
Nitsch
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Zurzolo
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D000818:Animals; D001692:Biological Transport; D002264:Carboxylic Acids / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D016764:Cell Polarity; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology; D037341:Fumonisins; D006028:Glycosphingolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D008148:Lovastatin / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D017475:Receptors, Nerve Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility / Q000187:drug effects; D013961:Thyroid Gland / Q000166:cytology* / Q000378:metabolism; D014162:Transfection; D047392:beta-Cyclodextrins
D002264:Carboxylic Acids; D003505:Cyclodextrins; D037341:Fumonisins; D006028:Glycosphingolipids; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D011506:Proteins; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C055324:phospholipase A2-activating protein; C056933:fumonisin B1; D017830:Octoxynol; D002784:Cholesterol; D008148:Lovastatin