Covi J A, Belote J M, Mykles D L
Department of Biology, Colorado State University, Fort Collins, Colorado, 80523, USA.
Arch Biochem Biophys. 1999 Aug 1;368(1):85-97. doi: 10.1006/abbi.1999.1294.
Two dominant temperature-sensitive (DTS) Drosophila mutants are missense mutations of proteasome genes encoding beta-type subunits beta6/C5 (DTS5) and beta2/Z (DTS7). At nonpermissive temperature (29 degrees C), heterozygotes (DTS5/+ and DTS7/+) develop normally until metamorphosis; pupae fail to mature and die before eclosion. Proteasomes were purified from wild-type (WT) and heterozygous adult flies raised at permissive temperature (25 degrees C). Two-dimensional gel electrophoresis separated at least 28 proteins, 13 of which were identified with monospecific antibodies to alpha6/C2 (five species), alpha2/C3 (three species), alpha7/C8 (three species), alpha5/zeta, and beta1/Y subunits. Both quantitative and qualitative differences were observed between WT and DTS/+ proteasomes, with DTS5/+ deviating more from WT than DTS7/+ proteasomes. In DTS5/+ there was a shift to more acidic species of C2 and C3 and a shift to less acidic species of 32-kDa subunits (#3-#7) recognized by an anti-alpha subunit monoclonal antibody (MCP222) and were losses of two 32-kDa subunits (#2 and #3), decreases in Y (25 kDa; 2-fold) and 31-kDa (#9; 2-fold) subunits, and increases in 52-kDa (#1; 1.9-fold) and 24-kDa (#13; 2.3-fold) subunits. In DTS7/+ there was a less pronounced shift to acidic species of C3 and no pI shift in C2 species and subunits #3-#7 and were decreases in #9 (2.5-fold) and #14 (3-fold) and a loss of #2. The three C8 species were similar between WT, DTS5/+, and DTS7/+ proteasomes. Qualitatively, the most dramatic difference was the appearance of a new 24-kDa subunit (#16) in DTS/+ preparations, with about a 14-fold greater amount of #16 in DTS7/+ than in DTS5/+ proteasomes. Catalytically, WT and DTS/+ proteasomes had similar peptidase activities, although the DTS/+ proteasomes were slightly more sensitive to SDS and elevated temperatures in vitro. The incorporation of DTS subunits apparently altered proteasome assembly and/or processing at permissive temperature with little effect on catalytic activities. These data suggest that at nonpermissive temperature, assembly/processing is more severely affected, producing DTS-containing complexes that lack functions essential for cellular proliferation and differentiation at metamorphosis.
两个显性温度敏感（DTS）果蝇突变体是蛋白酶体基因的错义突变，这些基因编码β型亚基β6/C5（DTS5）和β2/Z（DTS7）。在非允许温度（29摄氏度）下，杂合子（DTS5/+和DTS7/+）在变态前发育正常；蛹未能成熟并在羽化前死亡。蛋白酶体从在允许温度（25摄氏度）下饲养的野生型（WT）和杂合成年果蝇中纯化得到。二维凝胶电泳分离出至少28种蛋白质，其中13种通过针对α6/C2（5种）、α2/C3（3种）、α7/C8（3种）、α5/ζ和β1/Y亚基的单特异性抗体进行了鉴定。在WT和DTS/+蛋白酶体之间观察到了定量和定性的差异，DTS5/+比DTS7/+蛋白酶体偏离WT的程度更大。在DTS5/+中，C2和C3的物种向更酸性的方向转变，并且被抗α亚基单克隆抗体（MCP222）识别的32-kDa亚基（#3-#7）向酸性较弱的物种转变，并且丢失了两个32-kDa亚基（#2和#3），Y（25 kDa；2倍）和31-kDa（#9；2倍）亚基减少，52-kDa（#1；1.9倍）和24-kDa（#13；2.3倍）亚基增加。在DTS7/+中，C3向酸性物种的转变不太明显，C2物种以及亚基#3-#7没有pI偏移，并且#9（2.5倍）和#14（3倍）减少，#2丢失。WT、DTS5/+和DTS7/+蛋白酶体之间的三种C8物种相似。定性地说，最显著的差异是在DTS/+制剂中出现了一种新的24-kDa亚基（#16），DTS7/+中#16的量比DTS5/+蛋白酶体中的多约14倍。在催化方面，WT和DTS/+蛋白酶体具有相似的肽酶活性，尽管DTS/+蛋白酶体在体外对SDS和升高的温度稍微更敏感。DTS亚基的掺入显然在允许温度下改变了蛋白酶体的组装和/或加工，对催化活性影响很小。这些数据表明，在非允许温度下，组装/加工受到更严重的影响，产生了含有DTS的复合物，这些复合物缺乏变态时细胞增殖和分化所必需的功能。
