Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves
Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves
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DOI：
10.1016/j.plaphy.2003.12.004
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发表时间：
2004-03-01
影响因子：
6.5
通讯作者：
Huber, SC
中科院分区：
文献类型：
--
作者：
Hardin, SC;Huber, SC
玉米（Zea mays L.）蔗糖合成酶（SUS）（EC 2.4.1.13）与SUS蛋白稳定性的翻译后调节有关。为了阐明蛋白水解过程和磷酸化的作用，SUS降解和蛋白酶体活性在玉米叶片伸长区进行了研究。大小排阻色谱解析了蛋白酶体样蛋白水解活性的两个峰。大分子量（类似于1350 kDa）峰需要Mg 2+和ATP的最大活性，并抑制蛋白酶体抑制剂MG 132和NLVS。阴离子交换层析解决了类似的蛋白水解活性，激活ATP，特征是一致的26 S-蛋白酶体。适当地，免疫印迹揭示了存在的26 S-蛋白酶体亚基和高度泛素化的蛋白质内的活性组分从两个柱洗脱。较小分子量（类似于600 kDa）峰仅代表总蛋白酶体样活性的40%，可能是玉米20 S-蛋白酶体，因为其在体外被低水平十二烷基硫酸钠（SDS）激活。S170磷酸化的SUS（pS170-SUS）被检测为高分子量（HMM）形式和与26 S-蛋白酶体活性在尺寸排阻和阴离子交换柱上共洗脱的蛋白水解片段。保持最大26 S-蛋白酶体活性的条件降低了回收的pS170-SUS的量。在体外，26 S-蛋白酶体降解SUS和蛋白酶体特异性抑制剂减少SUS蛋白水解。HMM-SUS偶联物在体外产生，免疫沉淀表明，一些SUS可能在体内被泛素化。结果表明，S170磷酸化促进了HMM、泛在蛋白-SUS缀合物的形成，这些缀合物可以靶向26 S-蛋白酶体依赖性降解。(C)2004年由Elsevier SAS出版。
The serine-170 (S170) calcium-dependent protein kinase phosphorylation site of maize (Zea mays L.) sucrose synthase (SUS) (EC 2.4.1.13) has been implicated in the post-translational regulation of SUS protein stability. To clarify the proteolytic process and the role of phosphorylation, SUS degradation and proteasome activities were studied in the maize leaf elongation zone. Size-exclusion chromatography resolved two peaks of proteasome-like proteolytic activity. The large molecular mass ( similar to 1350 kDa) peak required Mg2+ and ATP for maximal activity and was inhibited by the proteasome inhibitors MG132 and NLVS. Anion-exchange chromatography resolved a similar proteolytic activity that was activated by ATP, characteristics that are consistent with those of a 26S-proteasome. Appropriately, immunoblotting revealed the presence of a 26S-proteasome subunit and highly ubiquitinated proteins within the active fractions eluted from both columns. The smaller molecular mass (similar to 600 kDa) peak represented only 40% of the total proteasome-like activity and is likely a maize 20S-proteasome as it was activated in vitro by low levels of sodium dodecyl sulfate (SDS). S 170 phosphorylated SUS (pS 170-SUS) was detected as both high molecular mass (HMM) forms and proteolytic fragments that co-eluted with 26S-proteasome activities on both size-exclusion and anion-exchange columns. Conditions that maintained maximal 26S-proteasome activity reduced the amounts of pS170-SUS recovered. In vitro, the 26S-proteasome degraded SUS and proteasome-specific inhibitors reduced SUS proteolysis. HMM-SUS conjugates were produced in vitro and immunoprecipitations suggested that some SUS might be ubiquitinated in vivo. The results suggest that S 170 phosphorylation promotes the formation of HMM, ubiquitin-SUS conjugates that can be targeted for 26S-proteasome-dependent degradation. (C) 2004 Published by Elsevier SAS.