Schröder S, Schimmöller F, Singer-Krüger B, Riezman H
Department of Biochemistry, Biozentrum, University of Basel, Switzerland.
J Cell Biol. 1995 Nov;131(4):895-912. doi: 10.1083/jcb.131.4.895.
The Saccharomyces cerevisiae EMP47 gene encodes a nonessential type-I transmembrane protein with sequence homology to a class of intracellular lectins defined by ERGIC-53 and VIP36. The 12-amino acid COOH-terminal cytoplasmic tail of Emp47p ends in the sequence KTKLL, which conforms with the consensus for di-lysine-based ER-localization signals. Despite the presence of this motif, Emp47p was shown to be a Golgi protein at steady-state. The di-lysine motif of Emp47p was functional when transplanted onto Ste2p, a plasma membrane protein, conferring ER localization. Nevertheless, the di-lysine motif was required for Golgi-localization of Emp47p and showed the same charge-independent, position-dependent characteristics of other di-lysine motifs. Alpha-COP has been shown to be required for ER localization of di-lysine-tagged proteins. Consistent with this finding, the Ste2p-Emp47p hybrid protein was mislocalized to the cell surface in the alpha-COP mutant, ret1-1. Surprisingly, the Golgi-localization of Emp47p was unaffected by the ret1-1 mutation. To investigate whether Emp47p undergoes retrograde transport from the Golgi to the ER like other di-lysine-tagged proteins we developed an assay to measure this step after block of forward transport in a sec12 mutant. Under these conditions retrograde transport led to a specific redistribution of Emp47p from the Golgi to the ER. This recycling occurred from a Golgi subcompartment containing alpha 1,3 mannose-modified oligosaccharides suggesting that it originated from a medial-or later Golgi compartment. Thus Emp47p cycles between the Golgi apparatus and the ER and requires a di-lysine motif for its alpha-COP-independent, steady state localization in the Golgi.
酿酒酵母EMP47基因编码一种非必需的I型跨膜蛋白，其序列与由ERGIC-53和VIP36定义的一类细胞内凝集素有同源性。Emp47p的12个氨基酸的COOH末端细胞质尾巴以KTKLL序列结束，这与基于双赖氨酸的内质网定位信号的共识相符。尽管存在这个基序，但在稳态时Emp47p被证明是一种高尔基体蛋白。当Emp47p的双赖氨酸基序移植到质膜蛋白Ste2p上时具有功能，赋予内质网定位。然而，双赖氨酸基序是Emp47p高尔基体定位所必需的，并且显示出与其他双赖氨酸基序相同的电荷无关、位置依赖的特征。已证明α-COP是双赖氨酸标记蛋白内质网定位所必需的。与这一发现一致，Ste2p-Emp47p杂合蛋白在α-COP突变体ret1-1中错误定位于细胞表面。令人惊讶的是，Emp47p的高尔基体定位不受ret1-1突变的影响。为了研究Emp47p是否像其他双赖氨酸标记蛋白一样从高尔基体进行逆向转运到内质网，我们开发了一种检测方法，用于在sec12突变体中阻断正向转运后测量这一步骤。在这些条件下，逆向转运导致Emp47p从高尔基体特异性重新分布到内质网。这种循环发生在含有α1,3甘露糖修饰寡糖的高尔基体亚区室，表明它起源于高尔基体中间或晚期区室。因此，Emp47p在高尔基体和内质网之间循环，并且其在高尔基体中α-COP非依赖性的稳态定位需要双赖氨酸基序。
