Expression, targeting, and assembly of functional Na,K-ATPase polypeptides in baculovirus-infected insect cells

A. W. DeTomaso, Jian Xie Zi Jian Xie, G. Liu, R. W. Mercer

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80 Scopus citations

Abstract

Deciphering the roles of the individual subunits of the heteromeric Na,K- ATPase in the structure, function, and assembly of this enzyme has been complicated because most expression systems have endogenous levels of Na,K- ATPase activity. This problem has become even more significant in light of the recent identification of multiple isoforms for both the α and β subunits. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, affords two distinct advantages for the expression of the Na,K-ATPase; some insect cells have little or no levels of Na,K-ATPase, and baculovirus-infected cells produce extremely high levels of foreign protein. We have made two separate recombinant baculoviruses containing the rodent α1 or β1 cDNAs and used them to infect the insect cell line Sf-9. The infected Sf-9 cells produce Na,K-ATPase subunit protein on the order of 5-10 μg of protein/ml of cultured cells. The rodent α1 polypeptide produced in the Sf-9 cells is indistinguishable electrophoretically and antigenically from the native subunit. The expressed β1 subunit is also antigenically identical but has a higher electrophoretic mobility due to differential glycosylation by the infected Sf-9 cell. In contrast to other systems, when expressed alone, each individual Na,K-ATPase subunit is targeted to the infected Sf-9 plasma membrane. In contrast, when infected with a virus that induces the heavy chain of murine IgG, the infected Sf-9 cell retains the polypeptide in the endoplasmic reticulum. However, when both IgG light and heavy chains are expressed, the polypeptides are properly processed and secreted. When the Na,K-ATPase α1 and β1 polypeptides are simultaneously expressed, they form detergent-resistant complexes that are functional. Ouabain-sensitive ATPase activity on the order of 5 μmol P(i)/mg/h in infected Sf-9 membranes was dependent on the expression of both the α1 and β1 subunits. Sodium- dependent phosphorylated intermediates were detected that were potassium- and ouabain-sensitive. No increase in ouabain-sensitive activity or phosphorylated intermediates was detected when either subunit was expressed alone. The α1β1-coinfected cells were also able to transport ions, as detected in 86Rb uptake experiments. Thus, the recombinant Na,K-ATPase expressed in insect cells is biologically active and is suitable for structural and functional analysis.

Original languageEnglish
Pages (from-to)1470-1478
Number of pages9
JournalJournal of Biological Chemistry
Volume268
Issue number2
StatePublished - 1993

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