FXYD7, Mapping of Functional Sites Involved in Endoplasmic Reticulum Export, Association With and Regulation of Na,K-ATPase

Gilles Crambert,Ciming Li,Lee Kim Swee,Käthi Geering

doi:10.1074/jbc.m313494200

Abstract

The brain-specific FXYD7 is a member of the recently defined FXYD family that associates with the alpha1-beta1 Na,K-ATPase isozyme and induces an about 2-fold decrease in its apparent K+ affinity. By using the Xenopus oocyte as an expression system, we have investigated the role of conserved and FXYD7-specific amino acids in the cellular routing of FXYD7 and in its association with and regulation of Na,K-ATPase. In contrast to FXYD2 and FXYD4, the studies on FXYD7 show that the conserved FXYD motif in the extracytoplasmic domain is not involved in the efficient association of FXYD7 with Na,K-ATPase. On the other hand, the conserved Gly40 and Gly29, located on the same face of the transmembrane helix, were found to be implicated both in the association with and the regulation of Na,K-ATPase. Mutational analysis of FXYD7-specific regions revealed the presence of an ER export signal at the end of the cytoplasmic tail. Deletion of a C-terminal valine residue in FXYD7 significantly delayed and decreased its O-glycosylation processing and retarded the rate of its cell surface expression. This result indicates that the C-terminal valine residue is involved in the rapid and selective ER export of FXYD7, which could explain the observed post-translational association of FXYD7 with Na,K-ATPase. In conclusion, our study on FXYD7 provides new information on structural determinants of general importance for FXYD protein action. Moreover, FXYD7 is identified as a new member of proteins with a regulated ER export, which suggests that, among FXYD proteins, FXYD7 has a particular regulatory function in brain.

Highlights

The Na,K-ATPase uses the hydrolysis of ATP to exchange Naϩ and Kϩ across the plasma membrane and to create and maintain the transmembrane Naϩ and Kϩ gradients
The brain-specific FXYD7 belongs to the FXYD protein family and was shown to associate with and regulate Na,K-ATPase transport properties [7]
FXYD7specific parts of the extracellular domain are important for its stability, conserved amino acids in extracellular and the transmembrane domain are involved in the association, probably of all FXYD proteins, with the Na,K-ATPase, and, the intracellular domain contains FXYD7-specific determinants involved in the intracellular routing of FXYD7

Summary

EXPERIMENTAL PROCEDURES

CDNAs—Mouse FXYD7 [7] served as a template for FXYD7 mutants (see Fig. 1). For truncated mutants (Nt-⌬15, Ct-⌬7, and ⌬V80), a series of single PCR reactions was carried out with appropriate primers. CRNAs coding for wild-type or mutant FXYD7 (2 ng/oocyte) were injected into oocytes in the presence or absence of cRNAs coding for the rat Na,KATPase ␣1 (10 ng/oocyte) and ␤1 (1 ng/oocyte) subunits as described in the figure legends. Under these conditions, wild-type, or mutant FXYD7 is expressed in excess of exogenous Na,K-ATPase and is not limiting for the association with Na,K-ATPase. After the pulse and chase periods, oocyte extracts were prepared as previously described [15] with a digitonin-containing buffer (0.5% w/v final concentration) and subjected to immunoprecipitations with FXYD7 antibodies [7] or Na,K-ATPase ␣-antibodies [16] under denaturing or non-denaturing conditions [15]. Nonspecific binding was determined on non-injected oocytes and subtracted from the total binding determined on cRNA-injected oocytes

RESULTS

TABLE I

DISCUSSION