Roth A F, Sullivan D M, Davis N G
Department of Surgery, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
J Cell Biol. 1998 Aug 24;142(4):949-61. doi: 10.1083/jcb.142.4.949.
The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode. Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G. Davis. 1996. J. Cell Biol. 134:661-674) and both depend on sequence elements within the receptor's regulatory, cytoplasmically disposed, COOH-terminal domain (CTD). Here, we concentrate on the Ste3p sequences required for constitutive endocytosis. Constitutive endocytosis is rapid. Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole where it is degraded, all with a t1/2 of 15 min. Deletion analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD that is the minimal sequence required for this rapid turnover. Deletions intruding into this interval block or severely slow the rate of endocytic turnover. Moreover, the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination. Not only necessary for ubiquitination and endocytosis, this sequence also is sufficient. When transplanted to a stable cell surface protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis and consequent vacuolar degradation. Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity-no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval. The high proportion of acidic and hydroxylated amino acid residues in this interval suggests a similarity to PEST sequences-a broad class of sequences which have been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and cytoplasmic proteins. A likely possibility, therefore, is that this sequence, responsible for both endocytosis and ubiquitination, may be first and foremost a ubiquitination signal. Finally, we present evidence suggesting that the true signal in the wild-type receptor extends beyond the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which extend another 21 residues to the COOH terminus of Ste3p. Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ubiquitination/endocytosis signal.
酵母a因子受体（由STE3编码）存在两种内吞作用模式，一种是依赖配体的内吞作用，另一种是组成型的、不依赖配体的模式。这两种模式都与受体泛素化有关（罗斯，A.F.，和N.G.戴维斯。1996。《细胞生物学杂志》134:661 - 674），并且都依赖于受体调节性的、位于细胞质的COOH末端结构域（CTD）内的序列元件。在这里，我们专注于组成型内吞作用所需的Ste3p序列。组成型内吞作用很快。受体被合成，运输到细胞表面，被内吞，然后运输到液泡中降解，整个过程的半衰期为15分钟。缺失分析确定了一个位于Ste3p CTD的COOH末端附近的36个残基长的序列，这是这种快速周转所需的最小序列。侵入这个区间的缺失会阻断或严重减缓内吞周转的速率。此外，相同的36个残基序列指导受体泛素化。缺失该序列的突变体显示出无法检测到的泛素化水平，而具有中等内吞缺陷的突变体显示出与之相关的泛素化水平降低。这个序列不仅对泛素化和内吞作用是必需的，也是充分的。当移植到一个稳定的细胞表面蛋白——质膜ATP酶Pma1p上时，36个残基的STE3信号指导PMA1 - STE3融合蛋白的泛素化、内吞作用以及随后的液泡降解。对36个残基长的区间进行丙氨酸扫描诱变突出了其整体复杂性——没有发现单一的序列基序或信号，相反，所需的序列元件分布在整个区间。这个区间中酸性和羟基化氨基酸残基的高比例表明它与PEST序列相似——这是一类广泛的序列，已被证明可指导短命的核蛋白和细胞质蛋白的泛素化及随后的蛋白酶体降解。因此，一种可能的情况是，这个负责内吞作用和泛素化的序列可能首先是一个泛素化信号。最后，我们提供的证据表明，野生型受体中的真正信号超出了被定义为最小信号的36个残基长的序列，还包括延伸到Ste3p的COOH末端另外21个残基的连续的类PEST序列。与在另外两种酵母质膜蛋白中鉴定出的序列一起，STE3序列定义了一类新的泛素化/内吞作用信号。
