Brown C R, Martin R L, Hansen W J, Beckmann R P, Welch W J
Department of Medicine, University of California, San Francisco 94143-0854.
J Cell Biol. 1993 Mar;120(5):1101-12. doi: 10.1083/jcb.120.5.1101.
Mammalian cells constitutively express a cytosolic and nuclear form of heat shock protein (hsp) 70, referred to here as hsp 73. In response to heat shock or other metabolic insults, increased expression of another cytosolic and nuclear form of hsp 70, hsp 72, is observed. The constitutively expressed hsp 73, and stress-inducible hsp 72, are highly related proteins. Still unclear, however, is exactly why most eukaryotic cells, in contrast to prokaryotic cells, express a novel form of hsp 70 (i.e., hsp 72) after experiencing stress. To address this question, we prepared antibodies specific to either hsp 72 or hsp 73 and have compared a number of biological properties of the two proteins, both in vivo and in vitro. Using metabolic pulse-chase labeling and immunoprecipitation analysis, both the hsp 72 and hsp 73 specific antibodies were found to coprecipitate a significant number of newly synthesized proteins. Such interactions appeared transient and sensitive to ATP. Consequently, we suspect that both hsp 72 and hsp 73 function as molecular chaperones, interacting transiently with nascent polypeptides. During the course of these studies, we routinely observed that antibodies specific to hsp 73 resulted in the coprecipitation of hsp 72. Similarly, antibodies specific to hsp 72 were capable of coprecipitating hsp 73. Using a number of different approaches, we show that the constitutively expressed, pre-existing hsp 73 rapidly forms a stable complex with the newly synthesized stress inducible hsp 72. As is demonstrated by double-label indirect immunofluorescence, both proteins exhibit a coincident locale within the cell. Moreover, injection of antibodies specific to hsp 73 into living cells effectively blocks the ability of both hsp 73 and hsp 72 to redistribute from the cytoplasm into the nucleus and nucleolus after heat shock. These results are discussed as they relate to the possible structure and function of the constitutive (hsp 73) and highly stress inducible (hsp 72) forms of hsp 70, both within the normal cell as well as in the cell experiencing stress.
哺乳动物细胞组成性表达一种胞质和核形式的热休克蛋白（hsp）70，本文中称为hsp 73。在热休克或其他代谢损伤刺激下，可观察到另一种胞质和核形式的hsp 70，即hsp 72的表达增加。组成性表达的hsp 73和应激诱导的hsp 72是高度相关的蛋白质。然而，目前仍不清楚的是，与原核细胞相比，为何大多数真核细胞在经历应激后会表达一种新形式的hsp 70（即hsp 72）。为了解决这个问题，我们制备了针对hsp 72或hsp 73的特异性抗体，并比较了这两种蛋白质在体内和体外的一些生物学特性。使用代谢脉冲追踪标记和免疫沉淀分析，发现hsp 72和hsp 73特异性抗体均能共沉淀大量新合成的蛋白质。这种相互作用似乎是短暂的且对ATP敏感。因此，我们推测hsp 72和hsp 73均作为分子伴侣发挥作用，与新生多肽短暂相互作用。在这些研究过程中，我们经常观察到hsp 73特异性抗体导致hsp 72共沉淀。同样，hsp 72特异性抗体也能够共沉淀hsp 73。使用多种不同方法，我们发现组成性表达的、预先存在的hsp 73能迅速与新合成的应激诱导型hsp 72形成稳定复合物。如双标记间接免疫荧光所示，这两种蛋白质在细胞内呈现重合的定位。此外，将hsp 73特异性抗体注射到活细胞中可有效阻断热休克后hsp 73和hsp 72从细胞质重新分布到细胞核和核仁的能力。本文将结合hsp 70的组成型（hsp 73）和高度应激诱导型（hsp 72）形式在正常细胞以及应激细胞中可能的结构和功能来讨论这些结果。
