Structures of the Carbohydrate Recognition Domain of Ca2+-independent Cargo Receptors Emp46p and Emp47p

Tadashi Satoh,Ken Sato,Akira Kanoh,Katsuko Yamashita,Yusuke Yamada,Noriyuki Igarashi,Ryuichi Kato,Akihiko Nakano,Soichi Wakatsuki

doi:10.1074/jbc.m512258200

Abstract

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.

Highlights

In eukaryotic cells, post-translational modification of secreted proteins and intracellular protein transport between organelles are ubiquitous features
It was shown that the putative ligandbinding site of p58/ER-Golgi intermediate compartment (ERGIC)-53 is similar to the mannose-binding site of the L-type lectins
To our surprise, we found that Emp46p carbohydrate recognition domains (CRD) bind another metal ion Kϩ bound in the crystal structures

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The DNA fragments for residues 6 –229 of CRD of Emp46p, 7–227 of CRD of Emp47p (⌬1-Emp47p CRD), and 1–254 of CRD of Emp47p of S. cerevisiae were cloned into the BamHI and SmaI sites of the pGEX4T-1 plasmid (Amersham Biosciences). Crystals of native and SeMet-substituted Emp46p CRD in its Kϩ-bound forms were obtained in a buffer containing 8 mg mlϪ1 protein, 20% (w/v) PEG3350, 0.3 M KF, 0.1 M HEPES-Na (pH 7.5), and 10% (v/v) ethylene glycol, with incubation at 289 K for 4 days. The crystal of the metal-free Emp46p CRD was obtained in a buffer containing 8 mg mlϪ1 protein, 22% (w/v) PEG1000, 0.1 M HEPES-Na (pH 7.5), and 10% (v/v) ethylene glycol, with incubation at 289 K for 4 days These native and SeMet-substituted proteins in the absence and presence of the Kϩ ion were crystallized in space group P21 with two molecules per asymmetric unit.

Total reflections

RESULTS

DISCUSSION

Water molecules