Recombinant Mouse Signal recognition particle receptor subunit beta (Srprb)

What is the molecular structure and function of mouse Srprb?

Mouse Srprb functions as the beta subunit of the signal recognition particle receptor (SR), a heterodimeric protein complex essential for cotranslational targeting of secretory and membrane proteins to the endoplasmic reticulum (ER). Structurally, Srprb is a 30-kD transmembrane protein that belongs to the GTPase superfamily, distantly related to ARF and Sar1 . The protein contains a guanine nucleotide-binding domain, suggesting its role extends beyond merely anchoring the SRα subunit to the ER membrane .

When investigating the structure-function relationship of mouse Srprb, researchers should consider:

• Expression systems that preserve post-translational modifications

• Membrane reconstitution methods to maintain native conformation

• GTP binding assays using UV cross-linking techniques to confirm functionality

• Proteolytic digestion experiments to analyze protein interactions

How do researchers effectively express and purify recombinant mouse Srprb?

To obtain functional recombinant mouse Srprb, consider the following methodological approach:

• Expression system selection: BL21(DE3*) cells have been successfully used for coexpression of SRα and SRβΔTM (transmembrane domain-deleted version) using pET28a-hSRα and pET15b-SRβΔTM vectors respectively .

• Purification strategy:

  • For full-length Srprb with transmembrane domain, detergent solubilization is necessary

  • For functional studies, co-purification with SRα is recommended as they form a functional heterodimer

  • Affinity tags can be introduced, but verify they don't interfere with GTPase activity

• Quality control checks:

  • GTP binding assay to confirm nucleotide binding capacity

  • SRP interaction assays to verify functional activity

  • Size exclusion chromatography to ensure proper complex formation with SRα

What experimental models are suitable for studying mouse Srprb function?

When selecting experimental models to study mouse Srprb, researchers should consider:

• In vitro reconstitution systems:

  • Wheat germ extract systems have been used successfully for in vitro translation reactions to study SRP-dependent targeting

  • Salt-washed, trypsin-digested microsomal membranes (TKRM) provide a suitable membrane environment

• Cell-based models:

  • Mouse cell lines with tagged Srprb for localization studies

  • CRISPR/Cas9-mediated gene editing to introduce specific mutations

• Quantification approaches:

  • Translocation efficiency can be calculated using the equation:
    %Translocation = 100 × PL/(PL + pPL)
    where PL and pPL are the integrated intensities for prolactin and preprolactin bands from autoradiography

How does the MoRF element in SRα interact with mouse Srprb during ribosome sensing?

Recent research has identified a molecular recognition feature (MoRF) element in the disordered linker domain of the mammalian SRP receptor that plays an essential role in sensing the ribosome during cotranslational protein targeting . The MoRF element is conserved among eukaryotes and functions to accelerate SRP-SR assembly in response to ribosome binding.

When investigating this interaction:

• Experimental approach:

  • Use mutations in the SR linker (e.g., SRdL, SRdR, SRdM) to analyze the effect on ribosome-induced stimulation of SRP-SR assembly

  • Measure stimulated GTPase reactions between SRP and SR in the presence and absence of 80S ribosome

  • Utilize pre–steady-state fluorescence measurements to track SRP–SR interaction kinetics

• Key findings to consider:

  • Loss of the MoRF in the SRP receptor largely abolishes the ability of the ribosome to activate SRP-SR assembly

  • Mutations in the MoRF element reduce ribosome-induced stimulation from approximately 25-fold to merely 3-fold

  • The MoRF element appears to functionally replace the essential GNRA tetraloop in bacterial SRP RNA, representing an evolutionary shift from RNA to protein-based regulation

What are the comparative differences in GTPase regulation between mouse Srprb and bacterial SR?

Mouse Srprb belongs to the GTPase superfamily, but its regulation differs significantly from bacterial systems:

• Evolutionary differences:

  • Mammalian SR is a heterodimer of SRα and SRβ subunits, whereas bacterial SR is a single protein

  • Eukaryotic SRα contains an X-domain that binds tightly to SRβ, connected to the NG-domain through an intrinsically disordered linker of about 200 residues

  • The MoRF element in mammalian SR functionally replaces the electrostatic tether provided by the bacterial 4.5S RNA during SRP–SR interaction

• GTPase activation mechanism:

  • In the mammalian system, the interaction between SRP and SR is accelerated approximately 100-fold by the 80S ribosome and 20-fold by the signal sequence

  • The stimulatory effect is mediated primarily through the MoRF element in the SR linker

  • Kinetic analyses reveal that for WT SR, complex formation rate constant (kcat/Km) increases approximately 25-fold in the presence of the ribosome, but only 3-fold for MoRF mutants

• Experimental considerations:

  • When comparing GTPase activity, use the reciprocally stimulated GTPase reaction between SRP and SR as a readout of their interaction

  • Pre–steady-state fluorescence measurements provide valuable kinetic data on complex assembly

  • Consider the contributions of both signal sequence and ribosome in experimental design

How do researchers effectively analyze Srprb-dependent cotranslational targeting in vitro?

To analyze Srprb-dependent cotranslational targeting, consider this methodology:

• Experimental setup:

  • Initiate in vitro translation reactions of a model protein (e.g., pPL in wheat germ extract) containing 35S-methionine

  • Within 3 minutes, add a mixture containing:

    • 30 nM SRP

    • Variable concentrations (0-100 nM) of wild-type or mutant SR

    • 0.5 eq/μl of salt-washed, trypsin-digested microsomal membrane (TKRM)

  • Allow reaction to proceed for 40 minutes before quenching with SDS-loading buffer

• Analysis:

  • Separate proteins by SDS-PAGE and visualize by autoradiography

  • Quantify translocation efficiency using the formula:
    %Translocation = 100 × PL/(PL + pPL)
    where PL and pPL represent prolactin and preprolactin bands

  • Compare results between wild-type and mutant SR proteins to assess functional impact

• Controls to include:

  • SR-independent translocation (no SR added)

  • GTPase-deficient SR mutants

  • Signal sequence-deficient substrates

What structural elements of mouse Srprb contribute to SRP-ribosome interactions during targeting?

Recent electron cryo-microscopy structures of SRP and SRP·SR in complex with the translating ribosome have revealed key structural insights:

• Interaction architecture:

  • SRβ (Srprb) is an integral membrane protein that likely mediates the membrane association of SRα

  • The interaction involves a cascade of three directly interacting GTPases: SRP54, SRα, and SRβ

  • In early targeting complexes, the SRP54 M-domain with bound signal sequence positions next to the NG domain that interacts with ribosomal proteins uL23 and uL29

• Regulatory elements:

  • The structure reveals eukaryotic-specific C-terminal regions and their interactions with the signal sequence as it emerges from the ribosome tunnel

  • The signal sequence becomes buried within the binding groove as part of the targeting mechanism

  • Srprb's GTPase activity likely plays a regulatory role beyond simple anchoring

• Research approaches:

  • Cryo-EM analysis of targeting complexes assembled with GTP analogs (e.g., GDP-AlFx or 5'-guanylyl imidodiphosphate)

  • Structure-guided mutagenesis to validate functional importance of specific residues

  • Comparative analysis between structures captured at different states of the targeting process

How do post-translational modifications affect mouse Srprb function?

While specific data on post-translational modifications (PTMs) of mouse Srprb is limited in the provided search results, researchers should consider:

• Potential PTM sites:

  • GTPases often contain phosphorylation sites that regulate nucleotide binding/hydrolysis

  • Membrane proteins may undergo glycosylation affecting stability and localization

  • The transmembrane domain may experience lipid modifications

• Functional consequences to investigate:

  • Effects on GTP binding and hydrolysis rates

  • Alterations in SRα interaction strength

  • Changes in membrane localization or topology

  • Impact on ribosome-induced SR activation

• Methodological approaches:

  • Mass spectrometry to identify and quantify PTMs

  • Site-directed mutagenesis of potential modification sites

  • In vitro enzymatic assays with modified and unmodified forms

  • Cellular localization studies under different conditions that might affect modification status