ISOLATION AND CHARACTERIZATION OF A 14.5-KDA TRICHLOROACETIC-ACID-SOLUBLE TRANSLATIONAL INHIBITOR PROTEIN FROM HUMAN MONOCYTES THAT IS UP-REGULATED UPON CELLULAR-DIFFERENTIATION

A trichloroacetic-acid-soluble 14.5-kDa protein (p14.5) has been isola ted from human mononuclear phagocytes (MNP) by a combination of trichl oroacetic acid extraction, preparative electrophoresis and hydrophobic affinity chromatography; five tryptic peptides were subjected to prot ein sequencing. The full-length cDNA of the protein was cloned and seq uenced from a lambda gt11 human liver Library. The cDNA showed a remar kable similarity to a rat protein preferentially expressed in hepatocy tes and renal tubular epithelial cells. The encoded protein is 137 ami no acids long and similar to members of a new hypothetical family of s mall proteins with presently unknown function, named YER057c/YJGF. Hum an recombinant p14.5 inhibits in vitro protein synthesis in a rabbit r eticulocyte lysate system. Unlike other inhibitors of protein synthesi s, p14.5 is not phosphorylated despite the presence of putative phosph orylation sites. The p14.5 mRNA is weakly expressed in freshly isolate d monocytes but is significantly upregulated when these monocytes are subjected to differentiation. This is also reflected by a differentiat ion-dependent increase in the protein concentration as demonstrated by immunoblots from cytosolic fractions and fluorescence-activated flow cytometry of permeabilized cells. A differentiation-dependent mRNA and protein expression of p14.5 is further suggested by the observation o f a low expression in a variety of liver and kidney tumor cells and a high expression in fully differentiated cells as assessed by immunohis tochemistry and northern blots. The highest mRNA expression was found in hepatocytes and renal distal tubular epithelial cells and only weak expression was found in other human tissues as evaluated by northern blot analysis. The preferential localization of the immunoreaction pro duct seemed to be cytoplasmatic but, in less differentiated cells, nuc lear labeling was occasionally visible. Immunoblotting of subcellular fractions confirmed these data. The high degree of evolutionary conser vation of p14.5, the considerable upregulation during cellular differe ntiation and its potential role as a translational inhibitor may refle ct an involvement in basic cellular mechanisms, e.g. a differentiation -dependent regulation of protein synthesis in hepatocytes, renal tubul ar epithelial cells, smooth muscle cells and MNP.