Amino-terminal sequence analysis of the embryo-specific Prot proteins

The protein products of the Prot gene are specific to the maize embryo where they accumulate to high levels during seed development (D. Schwartz, MGG 174:233). Characterization of these proteins with respect to solubility in saline solutions indicates that they belong to the globulin class of seed proteins (A.L. Kriz and D. Schwartz, Plant Physiol. 82:1069). Our previous studies on synthesis of the Prot proteins have shown that the polypeptide of higher molecular mass, PROT', is processed post-translationally to form the "mature" gene product, PROT. The conversion of PROT' to PROT is dependent on the product of the unlinked gene Mep, and embryos homozygous for the recessive mep allele accumulate PROT' rather than PROT The action of Mep is significant in that this product appears to be a protease exclusively specific for PROT' and, to our knowledge, this is the only protease specific for a seed protein for which a defective allele is available. Characterization of Mep activity would therefore be a significant contribution to the knowledge of protein processing in the plant seed. To determine if Mep processing occurs in the amino-terminal portion of PROT', we determined the N-terminal amino acid sequence (20 residues) of both PROT' and PROT. As shown below, these sequences are not homologous and we thus infer that the Mep product proteolytically cleaves PROT' to form PROT. Note that the sequence of PROT' does not extend far enough to determine overlap with PROT, so it is not possible at this time to determine the site of Mep processing. We are in the process of screening an embryo cDNA library with Prot-specific oligonucleotides in order to identify clones corresponding to Prot, and eventual DNA sequence analysis of these clones will allow us to determine the precise site of Mep cleavage.

Sequences of polypeptides specified by the Prot-I allele
 
PROT': NH2-XXX XXX Phe Glu Phe Leu Val Leu Met
  Arg Gln Gly Val Arg Met Tyr Lys Gln XXX
  Pro/Asn ...
PROT: NH2-XXX Ser Pro Glu Glu Ser Ser Glu Asp Asp
  Asp Tyr Gln Phe Lys Val Lys Gln Gln Pro ...

Alan L. Kriz


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