AUTHENTIC PROCESSING AND TARGETING OF ACTIVE MAIZE AUXIN-BINDING PROTEIN IN THE BACULOVIRUS EXPRESSION SYSTEM

The major auxin-binding protein (ABP1) from maize (Zea mays L.) has be en expressed in insect cells using the baculovirus expression system. The recombinant protein can be readily detected in total insect cell l ysates by Coomassie blue staining on sodium dodecyl sulfate-polyacryla mide gel electrophoresis (SDS-PACE). Our data suggest that ABP1 is pro cessed similarly in both insect cells and maize. The signal peptide is cleaved at the same position as in maize and the mature protein under goes tunicamycin-sensitive glycosylation, yielding a product with the same mobility on SDS-PACE as authentic maize ABP1. On immunoblots the expressed protein is recognized by anti-KDEL monoclonal antibodies. Im munofluorescence localization demonstrates that it is targeted to and retained in the endoplasmic reticulum of insect cells in accordance wi th its signal peptide and KDEL retention sequence. The expressed ABP1 also appears to be active, since extracts of insect cells expressing A BP1 contain a saturable high-affinity 1-naphthylacetic acid-binding si te, whereas no saturable auxin-binding activity is detected in extract s from control cells.