Martinez E, Jimenez M A, Seguí-Real B, Vandekerckhove J, Sandoval I V
Facultad de Ciencias, Consejo Superior de Investigaciones Científicas,Universidad Autonoma de Madrid, Cantoblanco, Spain.
J Mol Biol. 1997 Apr 18;267(5):1124-38. doi: 10.1006/jmbi.1997.0925.
To investigate the role of the 17 residues long presequence (p17) in the transport of the precursor of yeast API (pAPI) from the cytosol to the vacuole we have studied the effects of point mutations upon its conformation and on the process of transport. 1H NMR analysis of p17 indicates that in aqueous solution 26% of the molecules have the 4-12 segment folded into an helix. The hydrophobic environment provided by SDS micelles promotes the folding of 54% of the p17 molecules into a 5-16 amphipathic alpha-helix. Both Schiffer-Edmunson helical wheel analysis of segment 4-12 and residue hydrophobic moments calculated considering all possible side-chain orientations between 80 and 120 degrees, indicate the amphipathic character of the helixes assembled in water and detergent. Charge interactions between the dipole pairs N-Glu2Glu3 and C-Lys12Lys13 are essential for helix stability and condition pAPI transport. Substitution of either Pro2Pro3 or Lys2Lys3 for Glu2Glu3, results in moderate destabilization of the helix, decreases protein targeting to the vacuolar membrane and partly inhibits translocation of the protein to the vacuolar lumen. Replacement of either Pro12Pro13 or Glu12Glu13 for Lys12Lys13, causes a major disruption of the helix, decreases protein targeting and blocks completely the translocation of the protein to the vacuolar lumen. Replacement of Gly7 for Ile7, a substitution which is known to destabilize alpha-helixes in peptides and proteins as a result of the peptide bond to the solvent at Gly residues, produces similar effects as the substitutions for the K12K13 pair. The effects of Gly7 on helix stability and protein transport are partly reversed by introduction of Asp residues at positions 2 and 3 and Ala at position 4. Replacements such as Arg2 for Glu2, or Arg6 for Glu6, which change the net and local charges of the presequence without altering its conformation, have no effect on the protein transport. These results provide direct evidence of the involvement of the presequence in the transport of pAPI from the cytosol to the vacuole. They show that folding of the pAPI presequence is conditioned by the physical/chemical properties of the environment and is critical for targeting the protein to the vacuolar membrane and for its translocation to the vacuolar lumen.
为了研究17个氨基酸残基的前导序列（p17）在酵母API前体（pAPI）从细胞质转运至液泡过程中的作用，我们研究了点突变对其构象及转运过程的影响。对p17的1H NMR分析表明，在水溶液中，26%的分子其4 - 12片段折叠成螺旋结构。SDS胶束提供的疏水环境促使54%的p17分子折叠成5 - 16两亲性α-螺旋。对4 - 12片段进行的Schiffer-Edmunson螺旋轮分析以及考虑80至120度之间所有可能侧链取向计算得到的残基疏水矩，均表明在水和去污剂中组装的螺旋具有两亲性。偶极对N-Glu2Glu3和C-Lys12Lys13之间的电荷相互作用对螺旋稳定性及pAPI转运至关重要。用Pro2Pro3或Lys2Lys3替代Glu2Glu3，会导致螺旋适度不稳定，减少蛋白质靶向液泡膜，并部分抑制蛋白质转运至液泡腔。用Pro12Pro13或Glu12Glu13替代Lys12Lys13，会导致螺旋严重破坏，减少蛋白质靶向并完全阻断蛋白质转运至液泡腔。用Ile7替代Gly7，已知这种替代会因甘氨酸残基处肽键与溶剂的作用而使肽和蛋白质中的α-螺旋不稳定，产生与K12K13对替代类似的效果。在2和3位引入天冬氨酸残基以及在4位引入丙氨酸残基，可部分逆转Gly7对螺旋稳定性和蛋白质转运的影响。用Arg2替代Glu2或用Arg6替代Glu6等替代方式，虽改变了前导序列的净电荷和局部电荷但不改变其构象，对蛋白质转运无影响。这些结果为前导序列参与pAPI从细胞质到液泡的转运提供了直接证据。结果表明，pAPI前导序列的折叠受环境物理/化学性质的制约，对蛋白质靶向液泡膜及其转运至液泡腔至关重要。
