Retinol binding protein and transthyretin are secreted as a complex formed in the endoplasmic reticulum in HepG2 human hepatocarcinoma cells

Diana Bellovino, Takashi Morimoto, Francesca Tosetti, Sancia Gaetani

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Abstract

Retinol binding protein (RBP), the retinol-specific carrier, circulates in blood as a 1:1 complex with the homotetrameric protein transthyretin (TTR). Both RBP and TTR are synthesized and secreted by the hepatocyte. In this work we have demonstrated, using HepG2 cells as a model system, that the association between the two proteins occurs inside the cell before secretion. The intracellular complex was detected only when metabolically labeled cells were lysed under mild detergent conditions (1.5% octylglucoside), followed by immunoprecipitation and SDS-PAGE. Alternatively, the immunoprecipitates from unlabeled cells lysed with the same buffer were analyzed by Western blotting. This finding was confirmed using the cross-linking agent dithiobis(succinimidyl) propionate before cell lysis. Moreover, we found that in cells treated with brefeldin A to block the exit of proteins from the endoplasmic reticulum (ER), the complex was present in the microsomal fraction. Thus, we can conclude that the RBP-TTR complex is formed inside the cell, more precisely within the ER. As RBP and TTR both lack an ER retention signal, we considered the possible involvement of chaperones in RBP and TTR retention in the ER and in complex formation. We found that calnexin, an ER integral membrane protein which functions as a chaperone, coprecipitates with RBP and TTR when cell lysis and immunoprecipitation are performed under mild conditions (1% Triton X-100). This result strongly suggests that calnexin may be involved in RBP and TTR retention in the ER, in TTR tetramer assembly, and possibly in complex formation.

Original languageEnglish
Pages (from-to)77-83
Number of pages7
JournalExperimental Cell Research
Volume222
Issue number1
DOIs
Publication statusPublished - Jan 10 1996

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ASJC Scopus subject areas

  • Cell Biology

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