Gross M, Olin A, Hessefort S, Bender S
Department of Pathology, University of Chicago, Illinois 60637.
J Biol Chem. 1994 Sep 9;269(36):22738-48.
We have purified a soluble rabbit reticulocyte protein, previously termed the supernatant factor, that reverses the inhibition of protein synthesis in hemin-deficient lysate by promoting the inactivation of the hemin-controlled eIF-2 alpha kinase (HCR) mediating the effect of hemin deficiency. We have identified the supernatant factor as a member of the heat shock protein 70 family, denoted hsp 70(R), based upon its size (72 kDa), specific reaction to a monoclonal antibody against eukaryotic hsp 70, strong binding affinity for ATP, and endogenous ATPase activity. We have investigated the role of hsp 70(R) and hemin in the regulation of the activation of HCR from its latent precursor (ProHCR) and the translational control of protein synthesis in rabbit reticulocyte lysate. We find that autophosphorylation of Pro-HCR is reduced by about 75% by adding saturating hsp 70(R) and almost completely reduced by adding either saturating hemin or limiting hemin plus limiting hsp 70(R). In contrast, autophosphorylation of HCR, which is similar in magnitude to that of ProHCR, is unaffected by adding either saturating hsp 70(R), saturating hemin, or limiting amounts of both. The activation of HCR (measured by inhibition of protein synthesis) from isolated ProHCR is completely prevented by hsp 70(R) in the presence, but not absence, of dithiothreitol. This suppression appears to be due to the association of hsp 70(R) with ProHCR, since hsp 70(R) action is prevented by ATP/Mg2+ and because activation of HCR from less purified ProHCR, that has associated hsp 70(R), is suppressed by dithiothreitol alone. This association is confirmed by sucrose gradient centrifugation, which shows co-sedimentation of some hsp 70(R) with ProHCR following preincubation together that is prevented by ATP/Mg2+ and does not occur after conversion of ProHCR to HCR. Limiting hsp 70(R) reduces the concentration of hemin required to prevent activation of HCR from isolated ProHCR from 0.75 to 0.15 microM and the optimal hemin concentration needed to maintain protein synthesis in reticulocyte lysate from 25 to 10 microM. Limiting hsp 70(R) also allows the delayed addition of hemin to suppress activation of HCR from ProHCR and to reverse inhibition of protein synthesis in hemin deficient lysate. The association of hsp 70(R) with ProHCR also underlies the observation that much more protein is synthesized in reticulocyte lysate in the absence of hemin at 25 degrees C than at temperatures of 30 degrees C or greater. These observed effects may be specific to hsp 70(R), since they are not observed with rabbit reticulocyte eIF-2 or eIF-2B, and since the comparable hsp 70 from bovine brain is incapable of maintaining or restoring protein synthesis in hemin-deficient lysate.
我们已经纯化了一种可溶性兔网织红细胞蛋白，该蛋白先前被称为上清因子，它通过促进介导血红素缺乏效应的血红素控制的真核起始因子2α激酶（HCR）失活，来逆转血红素缺乏裂解物中蛋白质合成的抑制作用。基于其大小（72 kDa）、对针对真核热休克蛋白70的单克隆抗体的特异性反应、对ATP的强结合亲和力以及内源性ATP酶活性，我们已将上清因子鉴定为热休克蛋白70家族的成员，命名为hsp 70(R)。我们研究了hsp 70(R)和血红素在兔网织红细胞裂解物中HCR从其潜在前体（ProHCR）激活的调节以及蛋白质合成的翻译控制中的作用。我们发现，添加饱和量的hsp 70(R)可使Pro-HCR的自磷酸化降低约75%，而添加饱和量的血红素或限量的血红素加限量的hsp 70(R)则几乎可使其完全降低。相比之下，HCR的自磷酸化程度与ProHCR相似，添加饱和量的hsp 70(R)、饱和量的血红素或两者的限量均对其无影响。在存在二硫苏糖醇但不存在时，hsp 70(R)可完全阻止从分离的ProHCR激活HCR（通过抑制蛋白质合成来测量）。这种抑制作用似乎是由于hsp 70(R)与ProHCR的结合，因为ATP/Mg2+可阻止hsp 70(R)的作用，并且单独的二硫苏糖醇可抑制来自与hsp 70(R)相关的纯化程度较低的ProHCR激活HCR。蔗糖梯度离心证实了这种结合，其显示预孵育后一些hsp 70(R)与ProHCR共沉降，这被ATP/Mg2+阻止，并且在ProHCR转化为HCR后不会发生。限量的hsp 70(R)可将防止从分离的ProHCR激活HCR所需的血红素浓度从0.75 microM降至0.15 microM，并将维持网织红细胞裂解物中蛋白质合成所需的最佳血红素浓度从25 microM降至10 microM。限量的hsp 70(R)还允许延迟添加血红素以抑制ProHCR激活HCR并逆转血红素缺乏裂解物中蛋白质合成的抑制作用。hsp 70(R)与ProHCR的结合也是以下观察结果的基础，即在25℃下无血红素时，兔网织红细胞裂解物中合成的蛋白质比在30℃或更高温度下多得多。这些观察到的效应可能对hsp 70(R)具有特异性，因为在兔网织红细胞eIF-2或eIF-2B中未观察到这些效应，并且由于来自牛脑的可比hsp 70无法维持或恢复血红素缺乏裂解物中的蛋白质合成。
