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 been expressed in insect cells using the baculovirus expression system. The recombinant protein can be readily detected in total insect cell lysates by Coomassie blue staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our data suggest that ABP1 is processed similarly in both insect cells and maize. The signal peptide is cleaved at the same position as in maize and the mature protein undergoes tunicamycin-sensitive glycosylation, yielding a product with the same mobility on SDS-PAGE as authentic maize ABP1. On immunoblots the expressed protein is recognized by anti-KDEL monoclonal antibodies. Immunofluorescence localization demonstrates that it is targeted to and retained in the endoplasmic reticulum of insect cells in accordance with its signal peptide and KDEL retention sequence. The expressed ABP1 also appears to be active, since extracts of insect cells expressing ABP1 contain a saturable high-affinity 1-naphthylacetic acid-binding site, whereas no saturable auxin-binding activity is detected in extracts from control cells.
Macdonald
H
H
School of Biological Sciences, University of Bristol, United Kingdom.
Henderson
J
J
Napier
R M
RM
Venis
M A
MA
Hawes
C
C
Lazarus
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D007210:Indoleacetic Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010937:Plant Growth Regulators; D010940:Plant Proteins / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection; D003313:Zea mays / Q000378:metabolism*
D017931:DNA Primers; D007210:Indoleacetic Acids; D010937:Plant Growth Regulators; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C046131:auxin receptor, plant