Assembly of the extracellular domain of the Na,K-ATPase beta subunit with the alpha subunit. Analysis of beta subunit chimeras and carboxyl-terminal deletions.

M Hamrick,K.J Renaud,D.M Fambrough

doi:10.1016/s0021-9258(20)80535-3

M Hamrick, K.J Renaud + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(20)80535-3

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Abstract

The role of the extracellular domain of the Na,K-ATPase beta subunit in assembly with the alpha subunit was investigated. A chimeric protein consisting of the extracellular domain of the beta subunit fused with the transmembrane and cytoplasmic domains of dipeptidyl peptidase IV assembles with the alpha subunit. An inverse chimera consisting of the cytoplasmic and transmembrane domains of the beta subunit fused with the extracellular domain of dipeptidyl peptidase IV does not assemble with the alpha subunit. The assembly data from these chimeras demonstrate that the extracellular domain of the beta subunit is both necessary and sufficient for assembly with the alpha subunit. Deletions of up to 146 extracellular amino acids from the carboxyl terminus of the beta subunit appear to result in misfolding of the subunit, but do allow reduced assembly with the alpha subunit. Together, the assembly data from chimeras and carboxyl-terminal deletions have identified a 96-residue extracellular domain which contains sequences involved in subunit assembly. While the chimeric subunits properly localize to the plasma membrane, deletion of as few as 4 amino acids from the carboxyl terminus impairs the ability of the beta subunit to be transported to the plasma membrane.

Highlights

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Lish, that the xtracellular domain of the @ subunit is sufficient for assembly with the LY subunit. in thework presented here, we investigated the role of the p subunit extracellular domain
The Na,K-ATPase is a pIasma membrane protein that uses energy derived from ATP hydrolysis to in assembly with the LY subunit and in the ability of the p subunit to be transported to theplasma membrane

Summary

MATERIALS AND METHODS

CDNA C # ~ ~ r ~ ~ -cDANlAls for expression were subcloned into kDa) and a smaller glycosylated@ subunit (-40-60 kDa). The cDNAs encoding the chicken Na,K-ATPase cy subunit (22) and p1subunit (11)were subcloned into pBluescript as BamHI andEcoRI restriction fragments, respectively. After a freeze-thaw, extracts were centrifuged for 30 min (which includes sequences encoding part of the DPPIV extracellular at 110,000 X g to pellet debris, and the supernatant was added to 25 domain) were used to PCR-amplify a segment of the my@ cDNA. The resulting construct encodes anamino- were eluted from the beads directly into SDS-PAGE sample buffer terminally my-tagged chimera consisting of & cytoplasmic and trans- (62 mM Tris, pH 6.8, 10% glycerol, 3% SDS, 5% 2-mercaptoethanol) membrane domains (Ala2-SeP3)fused with the DPPIV extracellular without boiling. Yielding cDNAs that encode subunits with truncated carboxyl Cells were rinsed with buffer containing 1%bovine serum albumin termini. 5’-TCAGTTCTCCAGCCACTC-3(’C146)to PCR-amplify the mycp temperature with buffer containing 1%bovine serum albumin and cDNA.

RESULTS

B Chicken p c4

DISCUSSION