Nodulin-24 follows a novel pathway for integration into the peribacteroid membrane in soybean root nodules.
Nodulin-24 is a nodule-specific protein of the peribacteroid membrane (PBM) in soybean. It has an apparent molecular mass of 33 kDa while its full-length cDNA encodes a polypeptide of only 24 kDa. In vitro transcription of nodulin-24 cDNA followed by translation resulted in a peptide translocated into microsomal membranes with cleavage of a signal sequence. The cleavage site of the signal sequence in nodulin-24 was determined to be between Ala (A25) and Arg (R26) by microsequencing of the [3H]leucine-labeled processed peptide. Fusion of the signal sequence of nodulin-24 with the beta-glucuronidase peptide prevented co-translational cleavage of the signal sequence although the translocation of the fused protein into microsomes occurred co-translationally. Trypsin treatment of membrane-translocated nodulin-24 did not result in any alteration in size suggesting that the newly synthesized peptide is fully protected in the membrane vesicle. Fusion of nodulin-24 with beta-glucuronidase also showed no change in size following trypsin treatment, suggesting that nodulin-24 has no membrane-spanning region. In addition, in vitro synthesized nodulin-24 was present in the supernatant fraction after sonication of microsomal membranes. Mature nodulin-24, on the other hand, is not solubilized from PBM by sodium carbonate (pH 11) or EGTA and is soluble only in detergent. These data suggest that nodulin-24 is synthesized as a lumenal protein in the endoplasmic reticulum and post-translationally attached to the membranes en route to the PBM. This processing results in a significant increase in the apparent molecular mass of nodulin-24 which may be due to the attachment of membrane lipids as this protein shares characteristics with membrane lipoproteins of many pathogenic bacteria.
Cheon
C I
CI
Department of Molecular Genetics, Ohio State University, Columbus 43210.
Hong
Z
Z
Verma
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DP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D016384:Consensus Sequence; D005966:Glucuronidase / Q000096:biosynthesis / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D012231:Rhizobium / Q000378:metabolism*; D013025:Glycine max / Q000378:metabolism* / Q000382:microbiology; D014158:Transcription, Genetic
D008565:Membrane Proteins; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C045625:nodulin; D005966:Glucuronidase