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Vol. 19, Issue 12, 5579-5592, December 2008

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Oxidative Folding and Assembly with Transthyretin Are Sequential Events in the Biogenesis of Retinol Binding Protein in the Endoplasmic Reticulum

Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India

Submitted January 10, 2008; Revised September 2, 2008; Accepted September 11, 2008
Monitoring Editor: Jeffrey L. Brodsky

Retinol-binding protein (RBP) is secreted out of the cell in its ligand-bound holo-form. The apo-form of RBP is selectively retained within the endoplasmic reticulum (ER) by a mechanism that remains unknown. Using isolated microsomal system, we have recapitulated the biogenesis of RBP involving its oxidative folding and assembly with transthyretin in the ER. In addition to dissecting its pathway of disulfide oxidation, we have analyzed association of its early folding intermediates with ER-chaperones. Our results show that of the three intramolecular disulfides present in RBP (4–160, 70–174, and 120–129) the smallest loop (120–129) was most critical for RBP to fold. Its absence caused RBP to aggregate into an intermolecular disulfide-linked structure. After acquisition of the small loop, formation of one of the two big disulfides (4–160 or 70–174) was sufficient for RBP to acquire a folded state. Using cross-linking in intact microsomes and sedimentation on sucrose gradients, we show that newly synthesized RBP is associated with a complex of chaperones consisting of Grp94, BiP, PDI, and calnexin. The complex was constitutively present in the ER, independent of the presence of folding substrates. RBP dissociated from this complex coincident with the formation of one of the two big disulfide loops, whereas RBP mutant lacking both the large disulfides showed persistent association. While highlighting the matrix-like characteristics of ER in isolated microsomal system our results provide insight into RBP folding and assembly mechanisms that will aid our understanding of its complex secretion properties.

* These authors contributed equally to this work.

Address correspondence to: Utpal Tatu (tatu{at}biochem.iisc.ernet.in).

Abbreviations used: DMSO, dimethyl sulfoxide; DTT, dithiothreitol; GSSG, oxidized glutathione; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; RBP, retinol-binding protein; TTR, Transthyretin.