Hennessey J P, Scarborough G A
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1990 Jan 5;265(1):532-7.
Reconstituted proteoliposomes containing Neurospora plasma membrane H+-ATPase molecules oriented predominantly with their cytoplasmic portion facing outward have been used to determine the location of the NH2 and COOH termini of the H+-ATPase relative to the lipid bilayer. Treatment of the proteoliposomes with trypsin in the presence of the H+-ATPase ligands Mg2+, ATP, and vanadate produces approximately 97-, 95-, and 88-kDa truncated forms of the H+-ATPase similar to those already known to result from cleavage at Lys24, Lys36, and Arg73 at the NH2-terminal end of the molecule. These results establish that the NH2-terminal end of the H+-ATPase polypeptide chain is located on the cytoplasmic side of the membrane. Treatment of the same proteoliposome preparation with trypsin in the absence of ligands releases approximately 50 water-soluble peptides from the proteoliposomes. Separation of the released peptides by high performance liquid chromatography and spectral analysis of the purified peptides identified only a few peptides with the properties expected of a COOH-terminal, tryptic undecapeptide with the sequence SLEDFVVSLQR, and NH2-terminal amino acid sequence analysis identified this peptide among the possible candidates. Quantitative considerations indicate that this peptide must have come from H+-ATPase molecules oriented with their cytoplasmic portion facing outward, and could not have originated from a minor population of H+-ATPase molecules of reverse orientation. These results directly establish that the COOH-terminal end of the H+-ATPase is also located on the cytoplasmic side of the membrane. These findings are important for elucidating the topography of the membrane-bound H+-ATPase and are possibly relevant to the topography of other aspartyl-phosphoryl-enzyme intermediate ATPases as well.
重组蛋白脂质体含有主要以其胞质部分向外的方向排列的粗糙脉孢菌质膜H⁺-ATP酶分子，已被用于确定H⁺-ATP酶的NH₂和COOH末端相对于脂质双层的位置。在H⁺-ATP酶配体Mg²⁺、ATP和钒酸盐存在的情况下，用胰蛋白酶处理蛋白脂质体，会产生大约97 kDa、95 kDa和88 kDa的H⁺-ATP酶截短形式，类似于已知由分子NH₂末端的Lys24、Lys36和Arg73处的切割产生的那些形式。这些结果表明，H⁺-ATP酶多肽链的NH₂末端位于膜的胞质侧。在没有配体的情况下，用胰蛋白酶处理相同的蛋白脂质体制备物，会从蛋白脂质体中释放出大约50种水溶性肽。通过高效液相色谱分离释放的肽，并对纯化的肽进行光谱分析，仅鉴定出少数具有COOH末端胰蛋白酶十一肽预期特性的肽，其序列为SLEDFVVSLQR，并且NH₂末端氨基酸序列分析在可能的候选物中鉴定出了该肽。定量分析表明，该肽必定来自其胞质部分向外的H⁺-ATP酶分子，而不可能源自少数反向排列的H⁺-ATP酶分子。这些结果直接表明，H⁺-ATP酶的COOH末端也位于膜的胞质侧。这些发现对于阐明膜结合H⁺-ATP酶的拓扑结构很重要，并且可能也与其他天冬氨酰-磷酰化酶中间体ATP酶的拓扑结构有关。
