Béguin P, Wang X, Firsov D, Puoti A, Claeys D, Horisberger J D, Geering K
Institute of Pharmacology and Toxicology, University of Lausanne, Switzerland.
EMBO J. 1997 Jul 16;16(14):4250-60. doi: 10.1093/emboj/16.14.4250.
The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the gamma peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged gamma subunits in intact oocytes and protease protection assays show that the gamma peptide is a type I membrane protein lacking a signal sequence and exposing the N-terminus to the extracytoplasmic side. Co-expression of the rat or Xenopus gamma subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The gamma peptide does not influence the formation and cell surface expression of functional Na,K-ATPase alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. Gamma subunits do not associate with individual alpha or beta subunits but only interact with assembled, transport-competent alpha-beta complexes. Finally, electrophysiological measurements indicate that the gamma peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the gamma subunit of Na,K-ATPase.
与离子泵或通道相关的小的疏水性肽的作用仍知之甚少。通过使用非洲爪蟾卵母细胞作为表达系统，我们已对与钠钾ATP酶共同纯化的γ肽的结构和功能特性进行了表征。完整卵母细胞中表位标记的γ亚基的免疫放射性标记和蛋白酶保护试验表明，γ肽是一种I型膜蛋白，缺乏信号序列，其N端暴露于胞外侧。在卵母细胞中大鼠或非洲爪蟾γ亚基与各种蛋白质的共表达表明，它仅与钠钾ATP酶的同工酶特异性结合。γ肽不影响功能性钠钾ATP酶α-β复合物的形成和细胞表面表达。另一方面，γ肽本身需要与钠钾ATP酶结合，以便在卵母细胞中稳定表达并有效地转运到质膜。γ亚基不与单个α或β亚基结合，而是仅与组装好的、具有转运能力的α-β复合物相互作用。最后，电生理测量表明，γ肽调节钠钾泵的钾离子激活。这些数据首次证明了钠钾ATP酶γ亚基的膜拓扑结构、结合特异性和潜在的功能作用。
