Fast degradation of the auxiliary subunit of Na+/K+-ATPase in the plasma membrane of HeLa cells.
The cell-surface expression and function of multisubunit plasma membrane proteins are regulated via interactions between catalytic subunits and auxiliary subunits. Subunit assembly in the endoplasmic reticulum is required for the cell-surface expression of the enzyme, but little is known about subunit interactions once it reaches the plasma membrane. Here we performed highly quantitative analyses of the catalytic (alpha1) and auxiliary (beta1 and beta3) subunits of Na(+)/K(+)-ATPase in the HeLa cell plasma membrane using isoform-specific antibodies and a cell-surface protein labeling procedure. Our results indicate that although the beta-subunit is required for the cell-surface expression of the alpha-subunit, the plasma membrane contains more alpha-subunits than beta-subunits. Pulse-labeling and chasing of the cell-surface proteins revealed that degradation of the beta-subunits was much faster than that of the alpha1-subunit. Ubiquitylation, as well as endocytosis, was involved in the fast degradation of the beta1-subunit. Double knockdown of the beta1- and beta3-subunits by RNAi resulted in the disappearance of these beta-subunits but not the alpha1-subunit in the plasma membrane. All these results indicate that the alpha- and beta-subunits of Na(+)/K(+)-ATPase are assembled in the endoplasmic reticulum, but are disassembled in the plasma membrane and undergo different degradation processes.
Yoshimura
Shige H
SH
Graduate School of Biostudies, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto, 606-8502, Japan. yoshimura@lif.kyoto-u.ac.jp
Iwasaka
Shizuka
S
Schwarz
Wolfgang
W
Takeyasu
Kunio
K
D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013997:Time Factors
C503551:ATP1B1 protein, human; C519304:ATP1B3 protein, human; C499076:ATP1A1 protein, human; D000254:Sodium-Potassium-Exchanging ATPase