Thyroglobulin Is Selected as Luminal Protein Cargo for Apical Transport via Detergent-resistant Membranes in Epithelial Cells

Martı́n-Belmonte, Fernando; Alonso, Miguel A.; Zhang, Xiaoqing; Arvan, Peter

doi:10.1074/jbc.m005429200

Cited by 30 publications

(33 citation statements)

References 60 publications

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“…The partition of GPI-PLD, normally a secreted protein, in rafts was not due to the accumulation of the enzyme in the ER, since mature forms were also present in rafts. The presence of secreted proteins without transmembrane domains in rafts, such as GPI-PLD, has been reported previously [36]. GPI-PLD may localize in raft membranes through interaction with raft components such as its substrates, GPI anchor intermediates and GPI-anchored proteins, or lectins.…”

Section: Gpi-pld Is Active In Lipid Raftsmentioning

confidence: 99%

Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism

Mann

Hepworth

Raikwar

et al. 2004

Biochemical Journal

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GPI-PLD [glycosylphosphatidylinositol (GPI)-specific phospholipase D (PLD)] is a secreted mammalian enzyme that specifically cleaves GPI-anchored proteins. In addition, the enzyme has been shown to cleave GPI anchor intermediates in cell lysates. The biosynthesis of the GPI anchor is well characterized; however, the mechanisms by which the levels of GPI anchor intermediates are regulated are still unknown. To investigate whether GPI-PLD plays a role in this regulation, we isolated stable HeLa cells overexpressing the enzyme. GPI-PLD-HeLa (GPI-PLD-transfected HeLa) cells showed a 3-fold increase in intracellular GPI-PLD activity and drastically decreased the levels of GPI-anchored proteins when compared with untransfected HeLa controls. Intracellular cleavage of GPIanchored proteins has been suggested to occur early in the secretory pathway and, in agreement with this proposal, GPI-PLD activity in GPI-PLD-HeLa cells was detected not only in the endoplasmic reticulum and Golgi apparatus, but also in the plasma membrane. The enzyme was also active in lipid rafts, membrane microdomains in which GPI-anchored proteins and GPI anchor intermediates are concentrated, indicating that intracellular GPI-PLD cleavage may also occur in this compartment. Pulse-chase paradigms revealed the turnover rate of the last intermediate of the GPI anchor pathway in GPI-PLD-HeLa cells to be accelerated compared with the controls. Furthermore, 1,10-phenanthroline, a GPI-PLD inhibitor, reversed this effect. Our studies demonstrated that GPI-PLD can cleave not only GPIanchored proteins, but also GPI anchor intermediates intracellularly. This observation opens the possibility that GPI-PLD can influence the steady-state levels of GPI-anchored proteins by hydrolysing the anchor before and after its attachment to proteins.

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Section: Gpi-pld Is Active In Lipid Raftsmentioning

confidence: 99%

Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism

Mann

Hepworth

Raikwar

et al. 2004

Biochemical Journal

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“…It has been suggested that detergentresistant membranes function as apical targeting platforms (41). In addition it was recently shown that the apically targeted, secretory protein thyroglobulin associates with detergent-resistant membranes during transport to the cell membrane (42). However, a correlation between apical targeting and association to the detergent-resistant membrane could not be observed when wild-type serpins and chimeras of serpins were investigated.…”

Section: Discussionmentioning

confidence: 86%

Secretion of Antithrombin Is Converted from Nonpolarized to Apical by Exchanging Its Amino Terminus for That of Apically Secreted Family Members

Vogel¹,

Sahkri²,

Sjöström³

et al. 2002

Journal of Biological Chemistry

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The three members of the serpin family, corticosteroid binding globulin, ␣1-antitrypsin, and C1 inhibitor are secreted apically from Madin-Darby canine kidney (MDCK) cells, whereas two homologous family members, antithrombin and plasminogen activator inhibitor-1, are secreted in a nonpolarized fashion. cDNAs coding for chimeras composed of complementary portions of an apically targeted serpin and a nonsorted serpin were generated, expressed in MDCK cells, and the ratio between apical and basolateral secretion was analyzed. These experiments identified an amino-terminal sequence of corticosteroid binding globulin (residues 1-19) that is sufficient to direct a chimera with antithrombin mainly to the apical side. A deletion/mutagenesis analysis showed that no individual amino acid is absolutely required for the apical targeting ability of amino acids 1-30 of corticosteroid binding globulin. The corresponding amino-terminal sequences of ␣1-antitrypsin and C1 inhibitor were also sufficient to confer apical sorting. Based on our results we suggest that the apical targeting ability is encoded in the conformation of the protein.In epithelial cells, plasma membrane and secretory proteins are sorted in a polarized manner either to the apical or the basolateral surface. Sorting signals specifying basolateral sorting have been identified in the cytoplasmic domains of many membrane proteins (1, 2). Most of them are characterized either by essential tyrosine (3) or dileucine motifs (4). There is evidence that basolateral sorting of the receptors for transferrin and low density lipoproteins is mediated by AP-1 clathrin adaptors with an epithelia-specific isoform of subunit 1 (1B; Ref. 5). Much less is known about the signals and mechanisms of apical sorting. Apical signals appear to be localized to the noncytosolic segments of membrane proteins, since truncation mutants lacking the cytosolic and transmembrane portions were generally found to retain apical polarity (6 -9). Recently, a role of the transmembrane domains in apical sorting has been demonstrated for several proteins (10 -13). Also motifs in the cytoplasmic tails of rhodopsin (14) and the Na ϩ -dependent bile acid transporter (15, 16) have been shown to mediate apical sorting. With respect to polarized secretion of secretory proteins, it has been shown, for example, that MadinDarby canine kidney (MDCK) 1 cells secrete gp80/clusterin (17), erythropoietin (18), and corticosteroid binding globulin (CBG) (19) mainly from the apical side, whereas a range of proteins, including growth hormone, lysozyme, prochymosin, the immunoglobulin chain (20), cystatin C (6), and uteroglobin (21), are secreted equally from both plasma membrane domains. It was shown that N-linked glycans can act as apical targeting signals for secretory proteins, based on the observation that growth hormone, normally secreted in a nonpolarized manner from MDCK cells, is secreted mainly from the apical side after insertion of one or two N-linked glycosylation sites (22). In addition, erythropoietin, which...

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“…Raft Assay-Fractionation of Triton X-100 insoluble rafts was per-formed by standard procedure (32,33). Briefly, confluent cells pulselabeled for 30 min and chased for 1 h were rinsed twice with PBS and lysed for 20 min on ice in 1.4 ml of 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and 25 mM Tris-HCl, pH 7.5.…”

Section: Methodsmentioning

confidence: 99%

“…4). At this time, the labeled cells were extracted in buffer containing ice-cold 1% Triton X-100 and mixed with a small quantity of unlabeled cell extract containing caveolin-1 (see "Experimental Procedures"), a raft membrane marker (33). As shown in Fig.…”

Section: Fig 6 Trafficking Of Newly Synthesized Aat As Measured By mentioning

confidence: 99%

The Trafficking of α1-Antitrypsin, a Post-Golgi Secretory Pathway Marker, in INS-1 Pancreatic Beta Cells

Arvan²

2003

Journal of Biological Chemistry

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A sulfated ␣ 1 -antitrypsin (AAT), thought to be a default secretory pathway marker, is not stored in secretory granules when expressed in neuroendocrine PC12 cells. In search of a constitutive secretory pathway marker for pancreatic beta cells, we produced INS-1 cells stably expressing wild-type AAT. Because newly synthesized AAT arrives very rapidly in the Golgi complex, kinetics alone cannot resolve AAT release via distinct secretory pathways, although most AAT is secreted within a few hours and virtually none is stored in mature granules. Nevertheless, from pulse-chase analyses, a major fraction of newly synthesized AAT transiently exhibits secretogogue-stimulated exocytosis and localizes within immature secretory granules (ISGs). This trafficking occurs without detectable AAT polymerization or binding to lipid rafts. Remarkably, in a manner not requiring its glycans, all of the newly synthesized AAT is then removed from granules during their maturation, leading mostly to constitutive-like AAT secretion, whereas a smaller fraction (ϳ10%) goes on to lysosomes. Secretogogue-stimulated ISG exocytosis reroutes newly synthesized AAT directly into the medium and prevents its arrival in lysosomes. These data are most consistent with the idea that soluble AAT abundantly enters ISGs and then is efficiently relocated to the endosomal system, from which many molecules undergo constitutive-like secretion while a smaller fraction advances to lysosomes.

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Thyroglobulin Is Selected as Luminal Protein Cargo for Apical Transport via Detergent-resistant Membranes in Epithelial Cells

Cited by 30 publications

References 60 publications

Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism

Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism

Secretion of Antithrombin Is Converted from Nonpolarized to Apical by Exchanging Its Amino Terminus for That of Apically Secreted Family Members

The Trafficking of α1-Antitrypsin, a Post-Golgi Secretory Pathway Marker, in INS-1 Pancreatic Beta Cells

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