Shearer Alexander G, Hampton Randolph Y
University of California San Diego Division of Biological Sciences, Section of Cell and Molecular Biology, La Jolla, California 92093, USA.
J Biol Chem. 2004 Jan 2;279(1):188-96. doi: 10.1074/jbc.M307734200. Epub 2003 Oct 21.
The endoplasmic reticulum (ER) quality control pathway destroys misfolded and unassembled proteins in the ER. Most substrates of this ER-associated degradation (ERAD) pathway are constitutively targeted for destruction through recognition of poorly understood structural hallmarks of misfolding. However, the normal yeast ER membrane protein 3-hydroxy-3-methylglutaryl-CoA reductase (Hmg2p) undergoes ERAD that is physiologically regulated by sterol pathway signals. We have proposed that Hmg2p ERAD occurs by a regulated transition to an ERAD quality control substrate. Consistent with this, we had previously shown that Hmg2p is strongly stabilized by chemical chaperones such as glycerol, which stabilize misfolded proteins. To understand the features of Hmg2p that permit regulated ERAD, we have thoroughly characterized the effects of chemical chaperones on Hmg2p. These agents caused a reversible, immediate, direct change in Hmg2p degradation consistent with an effect on Hmg2p structure. We devised an in vitro limited proteolysis assay of Hmg2p in its native membranes. In vitro, chemical chaperones caused a dramatic, rapid change in Hmg2p structure to a less accessible form. As in the living cell, the in vitro action of chemical chaperones was highly specific for Hmg2p and completely reversible. To evaluate the physiological relevance of this model behavior, we used the limited proteolysis assay to examine the effects of changing in vivo degradation signals on Hmg2p structure. We found that changes similar to those observed with chemical chaperones were brought about by alteration of natural degradation signal. Thus, Hmg2p can undergo significant, reversible structural changes that are relevant to the physiological control of Hmg2p ERAD. These findings support the idea that Hmg2p regulation is brought about by regulated alteration of folding state. Considering the ubiquitous nature of quality control pathways in biology, it may be that this strategy of regulation is widespread.
内质网（ER）质量控制途径会降解内质网中错误折叠和未组装的蛋白质。这种内质网相关降解（ERAD）途径的大多数底物通过识别尚不明确的错误折叠结构特征而被持续靶向降解。然而，正常的酵母内质网膜蛋白3-羟基-3-甲基戊二酰辅酶A还原酶（Hmg2p）会经历由固醇途径信号进行生理调节的ERAD过程。我们曾提出，Hmg2p的ERAD是通过向ERAD质量控制底物的调节性转变而发生的。与此一致的是，我们之前已表明，Hmg2p会被诸如甘油等化学伴侣强烈稳定，这些化学伴侣能稳定错误折叠的蛋白质。为了解允许调节性ERAD的Hmg2p的特征，我们全面表征了化学伴侣对Hmg2p的影响。这些试剂导致Hmg2p降解发生可逆、即时、直接的变化，这与对Hmg2p结构的影响一致。我们设计了一种在其天然膜中对Hmg2p进行体外有限蛋白酶解分析的方法。在体外，化学伴侣使Hmg2p结构发生剧烈、快速的变化，变为一种较难接近的形式。如同在活细胞中一样，化学伴侣的体外作用对Hmg2p具有高度特异性且完全可逆。为评估这种模型行为的生理相关性，我们使用有限蛋白酶解分析来检测体内降解信号变化对Hmg2p结构的影响。我们发现，天然降解信号的改变会引发与化学伴侣作用时观察到的类似变化。因此，Hmg2p可经历与Hmg2p的ERAD生理控制相关的显著、可逆的结构变化。这些发现支持了Hmg2p调节是由折叠状态的调节性改变所导致的观点。鉴于质量控制途径在生物学中的普遍存在，这种调节策略可能很广泛。
