Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis
Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis
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通过电荷至丙氨酸扫描诱变获得的新型 CDC34 (UBC3) 泛素结合酶突变体
DOI：
10.1128/mcb.15.3.1210
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发表时间：
1995
影响因子：
5.3
通讯作者：
D. Gonda
中科院分区：
文献类型：
--
作者：
Z. Pitluk;M. McDonough;P. Sangan;D. Gonda
CDC 34（UBC 3）编码从芽殖酵母细胞周期的G1期向S期转变所需的泛素缀合（E2）酶。CDC 34由170个残基的催化N-末端结构域组成，其上附加有酸性C-末端结构域。细胞周期功能的一个可移动的决定簇位于C-末端结构域，但特定功能的决定簇也必须位于N-末端结构域。我们已经探索了“电荷-丙氨酸”扫描诱变的效用，以确定新的N-末端结构域突变体的CDC 34，是酶促能力相对于unfacilitated（E3-独立）泛素化，但尽管如此，是有缺陷的，其细胞周期功能。这些突变体可以揭示特定体内功能的决定因素，例如与底物相互作用或活性和底物选择性的反式作用调节剂所需的那些。18个“单扫描”突变体中的3个（其中带电残基的小簇突变为丙氨酸）在体内功能方面受到损害。一个突变体（cdc 34 -109，111，113 A）的目标是一个12残基的Cdc 34蛋白片段，在大多数其他E2中没有发现，并不能补充cdc 34无效突变体在低拷贝数，但可以补充无效突变体时，从诱导的GAL 1启动子过表达。将相邻的单扫描突变体组合以产生“双扫描”突变体产生了四个额外的突变体，其中两个显示出热敏感性和冷敏感性条件缺陷。在大肠杆菌中表达的突变体蛋白大多数表现出非易化性，（E3非依赖性）泛素缀合活性，但两个突变体与野生型和其他突变Cdc 34蛋白质的不同之处在于，它们在自泛素化反应-缀合酶功能期间催化的多泛素化程度，并且已经鉴定了另外的突变Cdc 34等位基因，这将在Cdc 34的进一步遗传和生物化学研究中有价值。依赖性泛素化
CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.
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泛素-蛋白质连接酶系统的蛋白质底物结合位点。
DOI：
--
发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Hershko,A;Heller,H;Eytan,E;Reiss,Y
通讯作者：
Reiss,Y
酵母细胞分裂周期基因 CDC4 和相关假基因的结构比较。
影响因子：
5.6
作者：
Yochem,J;Byers,B
通讯作者：
Byers,B
DOI：
--
发表时间：
1983-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
A. Hershko;H. Heller;S. Elias;A. Ciechanover
通讯作者：
A. Hershko;H. Heller;S. Elias;A. Ciechanover
影响因子：
2.9
作者：
GUAN, KL;DIXON, JE
通讯作者：
DIXON, JE
细菌表达的酵母CDC34基因产物可以进行自身泛素化，形成多泛素链连接蛋白。
DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Banerjee,A;Gregori,L;Xu,Y;Chau,V
通讯作者：
Chau,V