title: Stable interaction of the cargo receptor VIP36 with molecular chaperone BiP.
abstract: VIP36 is an intracellular lectin that cycles between the endoplasmic reticulum (ER) and the Golgi apparatus, and is thought to act as a cargo receptor in the transport and sorting of glycoproteins. Here we sought to identify the proteins that interact with VIP36 during the quality control of secretory proteins. VIP36 was crosslinked and immunoprecipitated from HEK293 cells that expressed Myc-tagged VIP36. An approximately 80 kDa protein coprecipitated with VIP36 and LC/MS/MS analysis revealed it to be immunoglobulin-binding protein (BiP), a major protein of the Hsp70 chaperone family. A VIP36 mutant with defective lectin activity was also proficient for the coimmunoprecipitation of an equivalent amount of BiP, indicating that the interaction between VIP36 and BiP was carbohydrate-independent. Immunoelectron microscopy experiment demonstrated that the interaction between VIP36 and BiP occurred in the ER. However, the VIP36 coprecipitated with BiP was resistant to endo beta-N-acetylglucosaminidase H treatment. A pulse-chase experiment revealed that the amount of BiP interacting with VIP36 did not change over more than 2 h. These results suggest that the interaction of VIP36 and BiP is not due to chaperone-substrate complex. Surface plasmon resonance analysis using recombinant proteins confirmed these binding characteristics of VIP36 and BiP in vitro. The interaction between recombinant soluble VIP36 and BiP is dependent on divalent cations but not on ATP. This mode of interaction is also different from that observed between BiP and its chaperone substrates. These observations suggest a new role for VIP36 in the quality control of secretory proteins.
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Nawa
authors.forename: Osamu
authors.initials: O
authors.lastname: Shimada
authors.forename: Norihito
authors.initials: N
authors.lastname: Kawasaki
authors.forename: Naoki
authors.initials: N
authors.lastname: Matsumoto
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D002412:Cations; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D006023:Glycoproteins / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000502:physiology*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000502:physiology*; D016253:Microscopy, Immunoelectron; D018832:Molecular Chaperones / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D002412:Cations; D000091342:Endoplasmic Reticulum Chaperone BiP; D006023:Glycoproteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C086797:LMAN2 protein, human; D037241:Mannose-Binding Lectins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase