DEVELOPMENTAL REGULATION OF A PLANT ENCODED INHIBITOR OF EUKARYOTIC INITIATION-FACTOR 2-ALPHA PHOSPHORYLATION

An inhibitor of eIF-2a phosphorylation was identified in various plant species. The plant protein (termed PKI) specifically cross-reacts wit h monoclonal antiserum that recognizes the glycosylated, active form o f a M-r 67 kD protein analog (p67) from reticulocytes. Northern blot a nalysis using a probe to the reticulocyte inhibitor cDNA further suppo rts the presence of analogous transcripts in plant tissue. PKI specifi cally inhibits the phosphorylation of the plant encoded eIF-2 alpha ki nase (pPKR) as well as plant and human eIF-2 alpha phosphorylation. Th e interaction between PKI and pPKR is indicated by their copurificatio n on dsRNA agarose, despite evidence showing that PKI does not bind ds RNA. Further, wheat PKI inhibits human PKR phosphorylation but activit y is recovered by immunodepletion of PKI from wheat germ fractions. PK I is temporally regulated during plant growth and development. It is m aximally present in extracts from dormant seeds, however, it is not de tectable soon after leaf emergence at approximately 48 h post-imbibiti on. PKI levels are again detectable at the mid-milk stage in seed deve lopment. Protein levels of pPKR in ribosomal salt wash and cytosolic e xtracts from healthy plant tissue remain essentially constant througho ut the life cycle. In contrast, pPKR activity levels based upon autoph osphorylation vary significantly and are inversely correlated with PKI protein levels. Phosphorylation of eIF-2 alpha is a classical mechani sm for the downregulation of protein synthesis suggesting that inhibit ion of pPKR activity by PKI may contribute to the dramatic and rapid i ncrease in protein synthesis observed during seed germination.