MTJ1/ERdj1 and its human homologue HTJ1 are membrane proteins that interact with the molecular chaperone BiP through their J-domain. HTJ1 also contains a C-terminal cytosolic region of Unknown function that consists of two SANT domains separated by a spacer region. We recently showed that the second SANT domain of HTJ1 (SANT2) binds to alpha 1-antichyrnotrypsin and alters its serpin activity [B. Kroczynska, C.M. Evangelista, S.S. Sainant, E-C. Elguindi, S.Y. Blond, The SANT2 domain of the murine tumor cell DnaJ-like protein I human homologue interacts with alpha 1-antichyrnotrypsin and kinetically interferes with its serpin inhibitory activity, J. Biol. Chem. 279 (2004) 11432-11443]. Here, we identified a new variant of human inter-or.-trypsin inhibitor heavy chain 4 (ITIH4) that also interacts with the SANT2 domain of HTJ1. Biochemical, mutagenesis, and fluorescence studies demonstrate that SANT2 binds to a carboxyl-terminal fragment that corresponds to the last third of the new ITIH4 isoform sequence (residues 588-930). ITIH4 and MTJ1 coimmunoprecipitate from total liver protein extracts and SANT2 protects the ITIH4588-930 recombinant fragment from being processed by kallikrein in vitro. This work reveals that the SANT2 domain of HTJ1 is a genuine protein-protein interaction module. (c) 2005 Elsevier Inc. All rights reserved.