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Abstract. Signal recognition particle (SRP) plays the key role in targeting secretory proteins to the membrane of the endoplasmic reticulum (Walter, P., and V. R. Lingappa. 1986. Annu. Rev. CeliBiol. 2:499-516). It consists of SRP7S RNA and six proteins. The 54-kD protein of SRP (SRP54) recognizes the signal sequence of nascent polypeptides. The 19-kD protein of SRP (SRP19) binds to SRP7S RNA directly and is required for the binding of SRP54 to the particle. We used deletion mutants of SRP19 and SRP54 and an in vitro assembly assay in the presence of SRP7S RNA to define the regions in both proteins which are required to form a ribonucleoprotein particle. Deletion of the 21 COOH-terminal amino acids of SRP19 does not interfere with its binding to SRP7S RNA. Further deletions abolish SRP19 binding to SRP7S RNA. The COOH-terminal 207 amino acids of SRP54 (M domain) were found to be necessary and sufficient for binding to the SRP19/7S RNA complex in vitro. Limited protease digestion of purified SRP confirmed our results for SRP54 from the in vitro binding assay. The SRP54M domain could also bind to Escherichia coli 4.5S RNA that is homologous to part of SRP7S RNA. We suggest that the methioninerich COOH terminus of SRP54 is a RNA binding domain and that SRP19 serves to establish a binding site for SRP54 on the SRP7S RNA.

Signal recognition particle (SRP) consists of a 7SL RNA molecule and 6 protein subunits. We have isolated and characterized cDNA clones from human liver which encode the 19kDa protein subunit (SRP19). This subunit binds to the RNA directly and mediates binding of a second polypeptide, the 54kDa subunit which is involved in signal sequence recognition. Amino acid sequences deduced from the human cDNA sequence were identical to amino acid sequences of tryptic peptides from canine pancreatic SRP19. In vitro transcription and translation of the human cDNA resulted in a protein product the same size as canine SRP19 which could be immunoprecipitated by an antiserum raised against canine SRP19. SRP19 synthesized in a cell-free system specifically bound to 7SL RNA. The sequence of SRP19 is discussed with respect to its binding to 7SL RNA.

In vitro, efficient translocation and glycosylation of the precursor of yeast alpha‐factor can take place post‐translationally. This property of prepro‐alpha‐factor appears to be unique as it could not be extended to other yeast protein precursors such as preinvertase or preprocarboxypeptidase Y. In order to determine if specific domains of prepro‐alpha‐factor were involved in post‐translational translocation, we carried out a series of experiments in which major domains were either deleted or fused onto reporter proteins. Fusion of various domains of prepro‐alpha‐factor onto the reporter protein alpha‐globin did not allow post‐translational translocation to occur in the yeast in vitro system. Prepro‐alpha‐factor retained its ability to be post‐translationally translocated when parts or all of the pro region were deleted. Removal of the C‐terminal repeats containing mature alpha‐factor had the most profound influence as post‐translational translocation decreased in proportion to the number of repeats deleted. Taken together, these results suggest that efficient post‐translational translocation requires a signal sequence and the four C‐terminal repeats. There does not however, appear to be specific information contained within the C‐terminus, as their presence in fusion did not enable the post‐translational translocation of reporter proteins. Lastly, the ability to post‐translationally translocate radiochemically pure prepro‐alpha‐factor that had been isolated by immuno‐affinity chromatography required the addition of a yeast lysate fraction. Moreover, post‐translational translocation is a function of the microsomal membrane of yeast microsomes and not of a factor peculiar to the yeast lysate, as reticulocyte lysate supported this as well.