Abstract
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.
Highlights
Tion ofnodulin-24cDNAfollowed bytranslation resulted Nodulin-26 isanintrinsicmembraneproteinandlacks a in a peptide translocated into microsomal membranes cleavable signal sequence (Miao et al, 1992)
The cleavagseite of strated that both amino and carboxyl ends of nodulin-26 face the signal sequence in nodulin-24 wasdetermined to be the hostcell cytoplasm (Miao et al, 1992)
Fusoifonthe suggested to be located on the surface facing the bacteroids signal sequence of nodulin-24 with the p-glucuronidase (Fortin et al, 1987)
Summary
In Vitro Dunslation andProcessing oftheProducts-In vitro synthesized transcripts were translated in a rabbit reticulocyte lysate using either[3"Slmethionine or [3H11eucine andcanine microsomal membranes (Promega Corp., Madison, WI). A nonionic detergent digested the processed nodulin-24 completely (Fig. 1, lane 4). The highest S(i) value assigned the cleavage site between Ala (A25) and Arg solved in a 10% SDS-polyacrylamide gel, and elutedby soaking in0.1% (R26). This sitealso satisfiesthe criteriaof the (-3, -1) rule for SDS. For experimental determination of the cleavage site, the [3H]leucine-labeled co-translationally processed product of nodulin-24 was subjected to Edman degradation.
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