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B. Hormone receptors

To complete our picture of the regulatory pathway of hormone-mediated gene expression, let's look at a putative auxin receptor in maize.

Hesse, T., Feldwisch, J., Balshüsemann, D., Bauw, G., Puype, M., Vandekerckhove, J., Löbler, M., Klämbt, D., Schell, J. and Palme, K. (1989) Molecular cloning and structural analysis of a gene from Zea mays (L.) coding for a putative receptor for the plant hormone auxin. The EMBO Journal vol. 8 no. 9 pp. 2453-2461.
 
STRATEGY:
 
Purify auxin-receptor protein
Amino acid analysis
Screen cDNA library with degenerate oligos
Sequence the gene
Analysis of expression:
RNA gel blots
Western blots of microsomal(3) proteins

 

1. Purification of auxin-receptor protein

Basic idea is to fractionate chromatographically and assay for binding activity.
 
Assay: 
protein extract
+ 3H NAA, 5min., 4°C
4.2M NHSO4
precipitated protein
assay in scintillation counter

 
 
 
Table 1. Purification of auxin-binding  proteins from Z. mays (L.) coleoptiles
Purification step Total protein
(mg.)		 Sp. act.
(pmol/mg)		 Total act.
(pmol)		 Yeild
(%)
Solubilized extract 237 114 27018 100
DEAE-Sephacel 132 82 10824 40
NAA-Sepharose 5.66 218 1234 4.6
MonoQ 0.52 1233 641 2.4

PURIFICATION OF AUXIN BINDING PROTEINS [Fig. 1]

A - elution profile
B - Coomassie staining
C - Western, using abp-specific antisera
Shows that purified protein represents a single polypeptide.
 

2. Amino acid analysis
 

PROTEIN SEQUENCE ANALYSIS OF AUXIN-BINDING PROTEIN [Table III]
 
Table 3. Protein sequence analysis of auxin-binding protein
Peptide Sequence
abp1 SxVRDNSLVRDISQMPQSSYGIEGLSHIT
abp2 sxPrdnslvrdisqxQqsNygGegFshit
abp3 SCVRDNSLVRDISQMPQxxYGEIGLSHIT
peptide 1 EVEVWLQTISPGQR
peptide 2a GTLLMGSSSL
peptide 2b FPFVWDEDCF

3. Screen cDNA library with degenerate oligos
 

Degenerate oligos:
 

17-mer from N-terminal region
26-mer from internal region
(sequences not reported in the paper!)


Screened maize cDNA library.

5 positive clones
4. Sequence the gene

Sequenced all 5 cloned inserts.

Clone paxrl, 840bp insert, agreed with aa. seq (shown in bold red)
 

NUCLEOTIDE AND AA SEQ. OF AUXIN BINDING PROTEIN ENCODING CDNA [Fig. 3] 
       -25        -15         -5 
CTCCGACATT CACGTGCAGC TGTCGGGAGC GGCA

                                                                             60 
ATG GCG CCG GAT CTA AGC GAA CTC GCC GCC GCC GCT GCA GCC CGT GGC GCC TAC CTC GCC 
M   A   P   D   L   S   E   L   A   A   A   A   A   A   R   G   A   Y   L   A   

                                                                            120 
GGC GTC GGT GTC GCG GTC CTC CTC GCT GCC TCC TTC CTC CCA GTA GCC GAG TCG TCC TGC 
G   V   G   V   A   V   L   L   A   A   S   F   L   P   V   A   E   S   S   C   

                                                                            180 
GTG CGA GAT AAC TCA TTG GTG AGA GAC ATA AGC CAA ATG CCG CAA AGC AGC TAT GGG ATT 
V   R   D   N   S   L   V   R   D   I   S   Q   M   P   Q   S   S   Y   G  I   

                                                                            240 
GAA GGA TTG TCA CAT ATA ACA GTT GCT GGT GCG CTC AAT CAT GGG ATG AAG GAG GTG GAA 
E   G   L   S   H   I   T   V   A   G   A   L   N   H   G   M   K   E   V   E   

                                                                            300 
GTG TGG CTT CAG ACA ATA AGT CCA GGT CAA AGG ACG CCG ATC CAC AGG CAT TCC TGT GAA 
V   W   L   Q   T   I   S   P   G   Q   R   T   P   I   H   R   H   S   C   E   

                                                                            360 
GAA GTT TTC ACT GTC CTC AAA GGG AAG GGT ACG CTC TTG ATG GGA TCA AGC TCA CTA AAG 
E   V   F   T   V   L   K   G   K   G   T   L   L   M   G   S   S   S   L   K   

                                                                            420 
TAC CCA GGG CAG CCA CAG GAA ATT CCT TTC TTT CAG AAT ACC ACA TTT TCA ATC CCT GTA 
Y   P   G   Q   P   Q   E   I   P   F   F   Q   N   T   T   F   S   I   P   V   

                                                                            480 
AAC GAT CCA CAC CAG GTT TGG AAT TCT GAC GAG CAC GAA GAT TTG CAA GTT CTT GTG ATC 
N   D   P   H   Q   V   W   N   S   D   E   H   E   D   L   Q   V   L   V   I   

                                                                            540 
ATT TCG AGA CCA CCT GCT AAG ATA TTT TTA TAT GAT GAT TGG AGC ATG CCT CAT ACA GCC 
I   S   R   P   P   A   K   I   F   L   Y   D   D   W   S   M   P   H   T   A   

                                                                            600 
GCG GTA CTG AAA TTC CCC TTC GTC TGG GAT GAG GAC TGC TTC GAA GCA GCA AAA GAC GAA 
A   V   L   K   F   P   F   V   W   D   E   D   C   F   E   A   A   K   D   E   


CTC TAG 
L   *   

                                                         700 
GTCACAAGTGTTTCCTGCAATGTATCTGCTTCATCCATGATCCTGCTGGACTACTAAAAT 

                                                         760 
TCACATGCACTAGTCGTAATAAAGCCAGTGCGCTTTTCATGTATAATTCTGTATTGTGGC 


TCGCTAAAATAAAATTTGGCAACGGTTTATG

leader seq. - 38aa's, mostly hydrophobic

N-glycosylation site - Asn-X-Thr/Ser; analysis of protein verified a branched mannan/N-acetyl glucosamine moiety
 

luminal signal - KDEL at C-term, typical of ER proteins; seems to be necessary for retention of proteins in lumen of ER.
 

5. Analysis of expression:
 

a) RNA gel blots
 
ORGAN-SPECIFIC EXPRESSION OF AUXIN-BINDING PROTEIN MRNA [Fig. 7]
 
 

mRNA level for abp in maize tissues, measured by densitometry of autoradiograms.  a: root; b: leaf; c: stem; d ears; e: style; f: tassel

Observed that auxins stimulate fruit setting. which is consistent with the highest concentration of axr mRNA being in maize ears.

b) Western blots of microsomal proteins
 
RELEASE OF AUXIN-BINDING PROTEINS FROM MICROSOMES [Fig. 8]
 
a - untreated microsomes, signal stays in pellet (p) after centrifugation.

Treatment with either b) triton X100 detergent or c) Na2CO3 disrupts membranes, releasing contents.

Authors note that during purification, auxin-binding activity partitions into aqueous phase, when extracted with Triton detergent. This suggests that the protein is not an integral membrane protein but rather is a normal constituent of the lumen of the ER.
 

microsomes - vesicles derived from disrupted ER during ER isolation.


Reem Aboukhaddour
Cooke TJ, Belle Poli D, Sztein AE, Cohen JD (2002) Evolutionary patterns in auxin action. Plant Mol. Biol. 49:319-338.
 
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