Abstract
KdpD is a four-spanning membrane protein that has two large cytoplasmic domains at the amino- and at the carboxyterminus, respectively. During its biogenesis KdpD binds to the signal recognition particle (SRP) of Escherichia coli that consists of a 48-kDa protein Ffh and a 4.5S RNA. The protein is targeted to the inner membrane surface and is released after contacting the SRP receptor protein FtsY. The information within the KdpD protein that confers SRP interaction was found in the amino-terminal cytoplasmic domain of KdpD, particularly at residues 22–48. Within this sequence a Walker A motif is present at residues 30–38. To determine the actual sequence specificity to SRP, a collection of mutants was constructed. When the KdpD peptides (residues 22–48) were fused to sfGFP the targeting to the membrane was observed by fluorescence microscopy. Further, nascent chains of KdpD bound to ribosomes were purified and their binding to SRP was analysed by microscale thermophoresis. We found that the amino acid residues R22, K24 and K26 are important for SRP interaction, whereas the residues G30, G34 and G36, essential for a functional Walker A motif, can be replaced with alanines without affecting the affinity to SRP-FtsY and membrane targeting.
Highlights
In Escherichia coli, the cytoplasmic signal recognition particle (SRP) mediates co-translational targeting of membrane proteins by binding to a “signal sequence” and generating a ribosome nascent chain complex (RNC)[1,2]
To verify the involvement of SRP in the membrane targeting of the N22-48-super folding GFP (sfGFP), we analysed the localization of the N22-48-sfGFP in the Ffh-depletion strain MC∆Ffh and found that SRP is required for membrane targeting of N22-48-sfGFP
The RNC-SRP complex is transported to the receptor protein FtsY to ensure that the nascent protein chain is close to the membrane surface
Summary
In Escherichia coli, the cytoplasmic signal recognition particle (SRP) mediates co-translational targeting of membrane proteins by binding to a “signal sequence” and generating a ribosome nascent chain complex (RNC)[1,2]. SRP at the ribosomal exit tunnel scans a nascent chain for bearing a hydrophobic SRP signal sequence[7] The presence of such an emerging SRP signal sequence causes a tight binding to the hydrophobic groove of the SRP M domain[8]. This RNC-SRP complex interacts with the membrane-associated SRP receptor, FtsY3. We investigated in detail the involvement of the Walker A motif and the positively charged residues in the signal sequence binding to SRP and the membrane targeting using sfGFP fusion proteins. When the positively charged residues outside the Walker box at positions [22, 24] and 26 are replaced by glutamines membrane targeting was inhibited
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