Kirisako T, Ichimura Y, Okada H, Kabeya Y, Mizushima N, Yoshimori T, Ohsumi M, Takao T, Noda T, Ohsumi Y
Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.
J Cell Biol. 2000 Oct 16;151(2):263-76. doi: 10.1083/jcb.151.2.263.
Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule "X" and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.
自噬和Cvt途径是通过双膜囊泡从细胞质到液泡的非经典囊泡运输的例子。Apg8/Aut7在这种囊泡的形成中起重要作用，尽管其具有亲水性，但它倾向于与膜结合。我们在此表明，Apg8与膜的结合性质会根据蛋白质本身的一系列修饰而改变。首先，新合成的Apg8的羧基末端精氨酸残基被一种新型半胱氨酸蛋白酶Apg4/Aut2去除，一个甘氨酸残基成为该蛋白质的羧基末端残基，现在被命名为Apg8FG。随后，Apg8FG与一个未鉴定的分子“X”形成共轭物，从而紧密结合到膜上。这种修饰需要Apg8FG的羧基末端甘氨酸残基和Apg7，一种泛素E1样酶。最后，通过Apg4的切割，加合物Apg8FG-X逆转回可溶性或松散结合于膜的Apg8FG。Apg4的作用方式，它既能切割新合成的Apg8，也能切割修饰后的Apg8FG，类似于去泛素化酶。第二次修饰可能涉及一种类似于泛素化的反应。Apg8的可逆修饰似乎与自噬和Cvt途径的膜动力学相关。
