Immunological evidence that plants use both HDEL and KDEL for targeting proteins to the endoplasmic reticulum.
The epitopes of two monoclonal antibodies raised to a putative auxin receptor have been mapped. Carboxy-peptidase A digestion of the antigen, auxin-binding protein (ABP) purified from maize, completely abolished binding of antibody MAC 256 and impaired binding of MAC 259, suggesting that they both recognise C-terminal epitopes. Published sequences of ABP showed that the C terminus was KDEL, a tetrapeptide used for targeting proteins to the ER in animal cells. We have used this short homology to confirm that the two monoclonals recognise C-terminal KDEL, showing that animal KDEL proteins and synthetic KDEL peptides are recognised and that animal cell ER is stained strongly and specifically. Sucrose density gradient fractionation of maize microsomal membranes showed that plant KDEL proteins, including ABP, fractionated with markers for the endoplasmic reticulum. However, few proteins are stained by anti-KDEL monoclonals in plants. For comparison, a monoclonal antibody raised to a synthetic HDEL peptide was also used and found to stain a set of proteins in all plant species tested. The anti-HDEL and anti-KDEL monoclonals were sequence specific, staining different proteins. On density gradient fractionation HDEL proteins also banded with ER marker activities. However, the intracellular distribution of HDEL and KDEL proteins determined by immunofluorescence was different. Whereas HDEL proteins showed a distribution characteristic of plant ER, and this localisation was confirmed by immunogold labelling of ultrathin sections and electron microscopy, KDEL proteins showed strong fluorescence in discrete parts of the cell cortex. These observations are discussed in terms of the potential these monoclonal antibodies have as markers for ER and of the role ABP plays in plant cell signalling.
Napier
R M
RM
Horticulture Research International, West Malling, Kent, UK.
Fowke
L C
LC
Hawes
C
C
Lewis
M
M
Pelham
H R
HR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001692:Biological Transport; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology / Q000378:metabolism*; D010937:Plant Growth Regulators; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D003313:Zea mays / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009842:Oligopeptides; D010937:Plant Growth Regulators; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; C046131:auxin receptor, plant; C073762:lysyl-aspartyl-glutamyl-leucine; C077480:histidyl-aspartyl-glutamyl-leucine