THE 24 KDA OUTER ENVELOPE MEMBRANE-PROTEIN FROM SPINACH-CHLOROPLASTS - MOLECULAR-CLONING, IN-VIVO EXPRESSION AND IMPORT PATHWAY OF A PROTEIN WITH UNUSUAL PROPERTIES

The 24 kDa outer envelope membrane protein of spinach chloroplasts (om p24) represents a major constituent of this membrane. Sequences of try ptic and endoprotease Glu-C peptides derived from omp24 allowed the de sign of oligonucleotides which were used to generate a DNA fragment by polymerase chain reaction using spinach cDNA as template. This fragme nt served as a probe to screen a cDNA library for a full-length clone of the omp24 coding sequence. The protein predicted from the complete sequence only has 148 amino acids and a molecular mass of 16294 Da. It is an acidic protein (calculated isoelectric point 4.8) with a high c ontent of proline residues. Expression of the coding sequence in Esche richia coli and characterization of the purified recombinant protein p roduced revealed that the overestimation of its molecular mass by SDS- PAGE (ca. 25 kDa) is due to its abnormal amino acid composition. Despi te its rather low hydrophobicity (polarity index 49%), omp24 appears t o be deeply embedded in the outer membrane. Insertion of omp24 into th e membrane proceeds almost independently of surface receptors or targe ting sequence but, in contrast to other known outer envelope membrane proteins, is stimulated by ATP.