Intersubunit Interactions in Human X,K-ATPases:  Role of Membrane Domains M9 and M10 in the Assembly Process and Association Efficiency of Human, Nongastric H,K-ATPase α Subunits (ATP1al1) with Known β Subunits

• Published in: Biochemistry (Easton) Vol. 39; no. 41; pp. 12688 - 12698
• Main Authors: Geering, Käthi, Crambert, Gilles, Yu, Chuliang, Korneenko, Tatyana V, Pestov, Nikolay B, Modyanov, Nikolai N
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.10.2000
• Subjects: Animals; Bufonidae; Female; Gastric Mucosa - enzymology; Gastric Mucosa - metabolism; Genetic Vectors - metabolism; H(+)-K(+)-Exchanging ATPase - biosynthesis; H(+)-K(+)-Exchanging ATPase - chemistry; H(+)-K(+)-Exchanging ATPase - genetics; H(+)-K(+)-Exchanging ATPase - metabolism; Humans; Hydrolysis; Membrane Proteins - biosynthesis; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Oocytes; Peptide Fragments - biosynthesis; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Processing, Post-Translational - genetics; Protein Structure, Tertiary - genetics; Rabbits; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Sodium-Potassium-Exchanging ATPase - biosynthesis; Sodium-Potassium-Exchanging ATPase - chemistry; Sodium-Potassium-Exchanging ATPase - genetics; Sodium-Potassium-Exchanging ATPase - metabolism; Trypsin; Xenopus
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0009791

Na,K- and H,K-ATPase (X,K-ATPase) α subunits need association with a β subunit for their maturation, but the authentic β subunit of nongastric H,K-ATPase α subunits has not been identified. To better define α−β interactions in these ATPases, we coexpressed human, nongastric H,K-ATPase α (AL1) and Na,K-ATPase α1 (α1NK) as well as AL1−α1 and α1−AL1 chimeras, which contain exchanged M9 and M10 membrane domains, together with each of the known β subunits in Xenopus oocytes and followed their resistance to cellular and proteolytic degradation and their ER exit. We show that all β subunits (gastric βHK, β1NK, β2NK, β3NK, or Bufo bladder β) can associate efficiently with α1NK, but only gastric βHK, β2NK, and Bufo bladder β can form stably expressed AL1−β complexes that can leave the ER. The trypsin resistance and the forces of subunit interaction, probed by detergent resistance, are lower for AL1−β complexes than for α1NK−β complexes. Furthermore, chimeric α1−AL1 can be stabilized by β subunits, but α1-AL1−gastric βHK complexes are retained in the ER. On the other hand, chimeric AL1−α1 cannot be stabilized by any β subunit. In conclusion, these results indicate that (1) none of the known β subunits is the real partner subunit of AL1 but an as yet unidentified, authentic β should have structural features resembling gastric βHK, β2NK, or Bufo bladder β and (2) β-mediated maturation of α subunits is a multistep process which depends on the membrane insertion properties of α subunits as well as on several discrete events of intersubunit interactions.