EXPERIMENTAL CELL RESEARCH 238, 273–282 (1998) ARTICLE NO. EX973860 Changes in the Expression of the Heme-Regulated eIF-2a Kinase and Heat Shock Proteins in Rabbit Reticulocytes Maturing during Recovery from Anemia 1 Sheri Uma, Donna J. Barret, 2 and Robert L. Matts 3 Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078-3035 response to heat stress, thus their nomenclature as Changes in the expression of the heme-regulated eIF- heat shock proteins (hsps) [4 – 8]. Subsequently hsps 2a kinase (HRI), heat shock proteins (Hsps, Hsp90, and were found to be induced by a number of other stress 70) and their associated cohorts (p60 and p23) were conditions including oxidative stress, heavy metals, studied in maturing rabbit reticulocytes during recov- amino acid analogs, nutrient deficiency, and pathogen ery from anemia. Reticulocytosis was induced by injec- infection [5, 9, 10]. In eukaryotes, hsps are coded for tion of N-acetylphenylhydrazine or by phlebotomy from by multigene families, some members of which are con- the ear vein, and circulating red blood cells were frac- stitutively expressed [5, 7, 11, 12]. Hsps expression is tionated on histopaque density gradients. Northern also regulated during development and cell differentia- and Western blot analyses indicated that HRI and hsps tion [13 – 17], suggesting that levels of hsps expression mRNA and protein content gradually decreased during might regulate cell differentiation [18 – 20]. maturation of reticulocytes into erythrocytes. Reduc- Molecular chaperones function in high molecular tion in levels of hsps and HRI was also observed when weight complexes referred to as chaperone machines cells of same age group (density) were compared as the [3]. Hsp90 and Hsp70 facilitate protein folding in con- animals recovered from the anemia. Low hematocrits junction with a number of cohorts: DnaJ-homologs; p48 correlated with high levels of hsps expression and with (Hip); p60 (Hop); p23; and a number of individual im- increasing hematocrits hsps expression decreased. Un- munophilins [21 – 28]. The Hsp90 chaperone machinery der the conditions used to quantify these protein levels, has been shown to function in maintenance of steroid Hsc70 and p60 were detected in erythrocytes of fully hormone receptors in a form competent to bind the recovered animals. Maintenance of Hsc70 and p60 sug- gests important ongoing roles for these hsps in pro- hormone [29, 30], in renaturation of denatured protein, tecting the structure and function of proteins in eryth- such as firefly luciferase [31, 32], in folding of the heme- rocytes lacking transcriptional and translational ma- regulated eIF-2a kinase (HRI) and p56 lck protein tyro- chinery.  1998 Academic Press sine kinase, and the maintenance of HRI in a conforma- tion that is competent to undergo activation [33, 34]. In reticulocytes, the heme-regulated eIF-2a kinase INTRODUCTION (HRI) coordinates the synthesis of globin with the availability of heme (reviewed in [35]). In addition to Molecular chaperones are ubiquitously expressed its activation in response to heme-deficiency, HRI is proteins which facilitate folding of newly synthesized activated under conditions that induce the heat shock proteins, stabilize proteins in partially unfolded states response (i.e., exposure to heat shock, heavy metal ions, for transport, facilitate the assembly and disassembly oxidants, and denatured proteins [36 – 39]). HRI inter- of multimeric protein complexes, prevent the aggrega- acts with Hsp90, Hsc70, FKBP52, and p23 [40–42]. tion of denatured proteins, and facilitate their renatu- The sensitivity of HRI to activation induced by stress ration [1 – 3]. The major protein families of molecular agents correlates inversely with the levels of Hsp70 chaperones were first identified as proteins induced in present in different preparations of RRL [43]. Rabbit reticulocyte lysate (RRL) is an important 1 This work was supported by Grant ES-04299 from the National model system for studying regulation of protein synthe- Institute of Environmental Health Sciences, NIH, and by the Okla- sis and it has become an important in vitro system for homa Agricultural Experiment Station (Project No. 1975). studying the mechanism and regulation of chaperone 2 Current address: Hybridon Corp., Worcester, MA 01604. function. However, different lysate preparations have 3 To whom correspondence and reprint requests should be ad- dressed. Fax: (405) 744-7799. E-mail: rlmatts@okway.okstate.edu. different characteristic protein synthesis ability and 273 0014-4827/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.