Disassembly and domain structure of the proteins in the proteins in the signal-recognition particle

Abstract

The signal-recognition particle (SRP) is a ribonucleoprotein (RNP) complex consisting of six different polypeptide chains and a 7SL RNA. It participates in initiating the translocation of proteins across the membrane of the endoplasmic reticulum, SRP was disassembled in 2 M KCl into three components, one RNP composed of 7SL RNA and the 54-kDa and 19-kDa proteins, and two heterodimers consisting of the 72/68-kDa and the 14/9-kDa proteins respectively. The 54-kDa protein could be released from the RNP subparticle by chromatography on DEAE-Sepharose in Mg2 ' -depleted buffer, while the 19-kDa protein remained bound to the 7SL RNA.The domain structure of SRP proteins was probed by using mild elastase treatment and protein-specific antibodies. It was found that the 72, 68, 54 and 19-kDa SRP proteins were proteolytically processed in distinct steps. Most remarkably a protein fragment of 55-kDa, generated from the 72-kDa SRP protein, and a 35-kDa fragment from the 54-kDa SRP protein were both released from the RNP particle. Fragments generated from the 68-kDa protein and detectable with the anti-(68-kDa protein) antibody remained associated with the RNP particle. Cleavage of the SRP proteins by elastase at 2.5 pg/ml resulted in partial loss of activity, while 10 lg/ml caused complete inactivation of the particle. Neither the elongation arrest of IgG light chain nor its translocation across SRP-depleted microsomal membranes was promoted. The implications of these results on the possible interaction between the SRP subunits are discussed.Secretory and membrane proteins are translocated across or inserted into the membrane of the endoplasmic reticulum (ER) [l]. This process is mediated by the signal-recognition particle (SRP) and docking protein, the receptor for SRP in the endoplasmic reticulum membrane [2 -51.Details of the functions of SRP and docking protein have been elucidated by using the wheat germ cell-free system and microsomal membranes derived from dog pancreas [4, 6, 71. It has been found that SRP interacts with the signal sequence in nascent secretory proteins and can arrest polypeptide chain elongation after about 70 and more amino acids have been polymerized [4,7, 81. When the arrested complex binds to the docking protein in the endoplasmic reticulum membrane, translation resumes and translocation of the nascent polypeptide chain across the membrane is initiated. According to these results, SRP would have at least three functional interactions: (a) recognition of nascent secretory polypeptides, (b) arresting elongation, and (c) interacting with the docking protein. This complex set of functions is probably reflected in the structural complexity of SRP.SRP is a rod-shaped RNP particle composed of six polypeptides with molecular masses of 72,68,54,19,14 and 9 kDa and one 7SL RNA molecule of 300 bases [9-111. The 7SL RNA can be divided into three distinct segments, the central 'S fragment', which contains a unique sequence of about 155 nucleotides, and the two flanking regions which show highCorrespondence ...

Characterization of the SRP68/72 interface of human signal recognition particle by systematic site‐directed mutagenesis

“…Conditions of even higher ionic strength (3 M KCl) than used earlier to isolate canine SRP68/72 yielded stable GST-72b 0 /Thx-H68e 0 complexes. 16 Attempts to distinguish between electrostatic and hydrophobic effects of this strong protein-protein interaction were unsuccessful. SRP68 and SRP72 could be separated by disruption of hydrogen bonds and unfolding using moderate concentrations of urea.…”

“…16 We incubated complexes between GST-72b 0 and Thx-H68e 0 at various potentially disruptive conditions, and measured the association of the polypeptides with Ni-NTA magnetic agarose beads. Figure 2(B) shows that the complex was stable at room temperature in buffers of elevated ionic strength (3 M KCl and 1 M LiCl, lanes 2 and 5), and remained intact in 20% ethanol and 5% DMSO (lanes 3 and 4).…”

“…22 Proteins SRP68 and SRP72 share the same phylogenetic distribution and are released from the SRP as a stable heterodimer. 10,16 We identified a stretch of 94 amino acid residues near the C-terminus of SRP68 (68e 0 ) which binds to $150 amino acids from the Nterminal region of SRP72 (72b 0 ). This 72b region is located within a predicted tandem array of four tetratricopeptide (TPR)-like motifs suggested to form a superhelical structure which binds to the C-terminal region of SRP68 by unknown means.…”

“…15 Nevertheless, in vitro experiments with elastase treated canine SRP demonstrated that the SRP68/P72 heterodimer contributes directly to the signal peptide recognition function in the assembled particle. 16 Recently, depletion of SRP68 and SRP72 by RNAi silencing was shown to be lethal for Trypanosoma brucei. 17 Transfection of HeLa cells with shRNA-expression plasmids directed against SRP72 led to a reduction of the SRP RNA levels.…”

Antibody to signal recognition particle in polymyositis

“…The signal recognition particle (SRP) is a complex of a unique RNA (labeled 7SL) (22,23) and 6 polypeptides with molecular weights of 72 kd, 68 kd, 54 kd, 19 kd, 14 kd, and 9 kd (24)(25)(26)(27). This cytoplasmic particle recognizes the signal sequence of secretory, lysosomal, and membrane proteins, binding to it via the 54-kd protein (28)(29)(30)(31)(32), and targets the nascent chains of these proteins to the endoplasmic reticulum by binding to a specific receptor (28)(29)(30)(31)(32)(33).…”