Lost in Translation: Regulation of Na Pump Subunit Abundance

Abstract

The Na pump is an αβ heterodimer responsible for maintaining fluid and electrolyte homeostasis in mammalian cells. We engineered MDCK cell lines expressing α1flag-, β1flag-, or β2myc-subunits via a tetracycline (tet)-regulated promoter and a cell line expressing both stable β1myc and tet-regulated β1flag in order to examine regulatory mechanisms of Na pump subunit expression. Overexpression of β1flag increases total β-subunit levels by greater than 200% without changes in α-subunit abundance, however, endogenous β1-subunit (β1E) abundance is decreased. β1E down-regulation does not occur during β2 overexpression. The decrease in β-subunit expression is not accompanied by any change in mRNA levels. In addition, the degradation rate of β-subunits is not altered by β1flag overexpression. Cells stably expressing β1myc, when induced to express β1flag-subunits show reduced β1myc- and β1E-subunit abundance, indicating that these effects occur via the coding sequences of the down-regulated polypeptides. Similarly, MDCK cells over-expressing α1flag-subunits exhibit a reduction of endogenous α1 (α1E) protein with no change in α mRNA levels or β-subunits. The reduction in α1E compensates for α1flag-subunit expression, resulting in unchanged total α-subunit abundance. Thus, regulation of α-subunit expression maintains its native level whereas β-subunit is not as tightly regulated and its abundance can increase substantially over native levels. These effects are also seen in HEK cells. This is the first indication that cellular Na pump subunit abundance is modulated by translational repression. We will discuss the mechanism of this novel, potentially important mode of Na pump regulation.