Nascent membrane and presecretory proteins synthesized in Escherichia coli associate with signal recognition particle and trigger factor

Abstract

SummaryThe Escherichia coli signal recognition particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system. In this study, we use an E. coli in vitro translation system in combination with bifunctional cross-linking reagents to investigate these interactions in more detail in a homologous environment. Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E. coli SRP. Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP. In addition, trigger factor is efficiently cross-linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E. coli ribosome.

Is Ffh required for export of secretory proteins?

“…It is, therefore, possible that many proteins move into the SecA/SecB pathway from SRP, and that these two pathways are not mutually exclusive. Consistent with this notion, PhoE utilizes a SecB-dependent pathway, yet the PhoE signal sequence has been found to crosslink with SRP upon readdition of wild-type lysate in a heterologous expression system [22] or when Ffh is added to physiological concentration in a homologous expression system [9]. In addition, some of the PhoA signal sequences, which are shown here to be sensitive to Ffh depletion, have also been shown to accumulate in the absence of SecB [12] and SecA [23] in vivo.…”

“…The substrate speci¢city of SRP for membrane proteins may re£ect the higher a¤nity of SRP for relatively hydrophobic signal sequences. Consistent with this idea, in vitro crosslinking studies have shown that more hydrophobic signal sequences are better crosslinked to Ffh [9,10]. Furthermore, crosslinking to trigger factor was unaffected by hydrophobicity [9].…”

“…These results also point to a limitation of Ffh-depletion experiments ; that is, complete depletion is rarely achievable and a residual amount of Ffh can be e¡ectively utilized by very e¤cient and hydrophobic signal sequences. Similarly, when SRP substrates are overproduced in a strain that is used to generate lysate, less Ffh is available for crosslinking to added nascent chains in vitro [9].…”

“…Consistent with this idea, in vitro crosslinking studies have shown that more hydrophobic signal sequences are better crosslinked to Ffh [9,10]. Furthermore, crosslinking to trigger factor was unaffected by hydrophobicity [9]. These studies utilized a series of signal peptides which varied in the ratio of alanine and leucine residues in the hydrophobic core, thereby varying the overall hydrophobicity in a systematic way.…”

SecYEG Proteoliposomes Catalyze the Δϕ-Dependent Membrane Insertion of FtsQ

“…Strain MC4100 was used to obtain S135 lysate. Plasmid pBSKftsQ was constructed by ligating the BamHI/HindIII fragment of pNB1 (21) into BamHI/HindIII-cut pC4meth101FtsQ (22).…”