Department of Biochemistry and Molecular Biology, The University of Georgia , Athens, Georgia 30602, United States.
Biochemistry. 2013 Sep 24;52(38):6601-14. doi: 10.1021/bi400647c. Epub 2013 Sep 10.
The Ras converting enzyme (Rce1p) is an endoprotease that is involved in the post-translational processing of the Ras GTPases and other isoprenylated proteins. Its role in Ras biosynthesis marks Rce1p as an anticancer target. By assessing the chemical accessibility of cysteine residues substituted throughout the Saccharomyces cerevisiae Rce1p sequence, we have determined that yeast Rce1p has eight segments that are protected from chemical modification. Notably, the three residues that are essential for yeast Rce1p function (E156, H194, and H248) are all chemically inaccessible and associated with separate protected segments. By specifically assessing the chemical reactivity and glycosylation potential of the NH2 and COOH termini of Rce1p, we further demonstrate that Rce1p has an odd number of transmembrane spans. Substantial evidence that the most NH2-terminal segment functions as a transmembrane segment with the extreme NH2 terminus projecting into the endoplasmic reticulum (ER) lumen is presented. Because each of the remaining seven segments is too short to contain two spans and is flanked by chemically reactive positions, we infer that these segments are not transmembrane segments but rather represent compact structural features and/or hydrophobic loops that penetrate but do not fully span the bilayer (i.e., re-entrant helices). We thus propose a topological model in which yeast Rce1p contains a single transmembrane helix localized at its extreme NH2 terminus and one or more re-entrant helices and/or compact structural domains that populate the cytosolic face of the ER membrane. Lastly, we demonstrate that the natural cysteine residues of Rce1p are chemically inaccessible and fully dispensable for in vivo enzyme activity, formally eliminating the possibility of a cysteine-based enzymatic mechanism for this protease.
Ras 转化酶 (Rce1p) 是一种内切蛋白酶，参与 Ras GTP 酶和其他异戊二烯化蛋白的翻译后加工。其在 Ras 生物合成中的作用标志着 Rce1p 是一种抗癌靶标。通过评估整个酿酒酵母 Rce1p 序列中取代的半胱氨酸残基的化学可及性，我们确定酵母 Rce1p 有八个区段受到化学修饰的保护。值得注意的是，对于酵母 Rce1p 功能至关重要的三个残基（E156、H194 和 H248）均无法进行化学修饰，并且与单独的保护区段相关。通过专门评估 Rce1p 的 NH2 和 COOH 末端的化学反应性和糖基化潜力，我们进一步证明 Rce1p 具有奇数个跨膜跨度。大量证据表明，最 NH2 末端的区段作为跨膜区段发挥作用，其极端的 NH2 末端突入内质网 (ER) 腔中。因为其余的七个区段中的每一个都太短而不能包含两个跨度，并且被具有化学反应性的位置所包围，所以我们推断这些区段不是跨膜区段，而是代表紧凑的结构特征和/或疏水环，它们穿透但不完全跨越双层（即，再进入螺旋）。因此，我们提出了一个拓扑模型，其中酿酒酵母 Rce1p 包含一个位于其极端 NH2 末端的单一跨膜螺旋和一个或多个再进入螺旋和/或紧凑的结构域，这些结构域填充 ER 膜的胞质侧。最后，我们证明 Rce1p 的天然半胱氨酸残基无法进行化学修饰，并且对体内酶活性完全不可或缺，这正式排除了该蛋白酶基于半胱氨酸的酶促机制的可能性。
