Protein SRP54 of human signal recognition particle: cloning, expression, and comparative analysis of functional sites
Introduction
All cells depend critically on the targeting of proteins to the proper compartment. The signal recognition particle (SRP) is a ribonucleoprotein complex that assures the co-translational insertion of secretory proteins into and through lipid bilayers (reviewed by Lütcke, 1995). SRP-mediated protein translocation was demonstrated first in a mammalian system derived from dog pancreas (Walter and Blobel, 1981a; Walter and Blobel, 1981b; Walter et al., 1981). Later, components of the SRP were identified in a wide variety of species (Zwieb and Larsen, 1997), indicating that SRPs are present in all organisms. The SRP contains one RNA molecule and up to seven different polypeptides (Walter and Blobel, 1983; Brown et al., 1994). Of the SRP proteins, SRP54 is always present and, with SRP RNA, is the sole constituent of the bacterial particle (Luirink and Dobberstein, 1994).
SRP interacts not only with the nascent signal peptide, but also with components of the membrane. The dynamic character of the mammalian SRP is underscored by its ability to adopt flexible RNA conformations (Zwieb and Ullu, 1986; Zwieb, 1989). For example, the cooperative association of human protein SRP19 with SRP RNA was shown to be mediated by an RNA conformational change (Walker et al., 1995) that was required for the interaction of protein SRP54 and SRP RNA (Gowda et al., 1997). Furthermore, protein SRP54, when bound to a signal peptide, could dissociate from the RNA, with the signal peptide being released upon GTP hydrolysis (Bacher et al., 1996).
Low temperature electron microscopic reconstruction of SRP54 in 3D (Czarnota et al., 1994) provided a visual representation of its two-domain character. In agreement with this view, the mild proteolytic digestion of protein SRP54 yielded the G-domain (SRP54G, a GTPase of known structure: Freymann et al., 1997), and the M-domain (SRP54M) (Römisch et al., 1990; Zopf et al., 1990). The SRP54M portion was named for its unusually high content of methionine residues, and is of particular interest as it interacts with the signal peptide, but also with the SRP RNA (Römisch et al., 1990; Zopf et al., 1990; Samuelsson, 1992; Zopf et al., 1993). It has remained a mystery how both functions are contained within a single, rather small domain. In addition, we do not understand the molecular mechanism by which a wide variety of signal peptides are recognized and targeted to the proper cellular compartment.
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cDNA cloning of human SRP54
An NcoI/EcoRI-fragment (467 bp) that corresponds to the 5′-portion of mouse SRP54 was isolated from plasmid SRP54-sac14 (Bernstein et al., 1989). The DNA was labeled radioactively with [α32P]dCTP using random priming with the Klenow fragment of DNA polymerase (Life Technologies, Grand Island, NY), and used to screen approximately 3×106 plaques of a λ-gt10 cDNA library of human HepG2 hepatoma cells obtained from the American Type Culture Collection, Rockville, Maryland (ATCC 77400). Plaques
Cloning and expression of human SRP54
Screening of a human cDNA library with a mouse SRP54 probe (Section 2) yielded a full-length cDNA clone (2017 bp) with an open reading frame for a polypeptide of 504 amino acids (GenBank Accession U51920). The calculated molecular weight of the human SRP54 protein was 55 704 Da. The deduced polypeptide sequence of human SRP54 was identical to that of canine SRP54 (Römisch et al., 1989), thus confirming the highly conserved nature of this protein. There were only two amino acid residue differences
Acknowledgements
We thank K. Ajay Sharma for help with recombinant protein expression in insect cells, Kimberly Chittenden and Kerfoot P. Walker III for expert technical assistance, and Martin Wiedmann for advice in the photoaffinity labeling with nascent signal peptides. This work was supported by NIH grant GM-49034 to C.Z.
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