Translation elongation regulates substrate selection by the signal recognition particle

Abstract

The signal recognition particle (SRP) is a universally conserved cellular machinery responsible for delivering membrane and secretory proteins to the proper cellular destination. The precise mechanism by which fidelity is achieved by the SRP pathway within the in vivo environment is yet to be understood. Previous studies have focused on the SRP pathway in isolation. Here we describe another important factor that modulates substrate selection by the SRP pathway: the ongoing synthesis of the nascent polypeptide chain by the ribosome. A slower translation elongation rate rescues the targeting defect of substrate proteins bearing mutant, suboptimal signal sequences both in vitro and in vivo. Consistent with a kinetic origin of this effect, similar rescue of protein targeting was also observed with mutant SRP receptors or SRP RNAs that specifically compromise the kinetics of SRP-receptor interaction during protein targeting. These data are consistent with a model in which ongoing protein translation is in constant kinetic competition with the targeting of the nascent proteins by the SRP and provides an important factor to regulate the fidelity of substrate selection by the SRP.

FIGURE 1.

Use of β-OH-Leu to probe the fidelity of protein translocation. A, incorporation of β-OH-Leu into nascent polypeptide reduces the efficiency of pPL translocation. B, the translocation defect of proteins containing β-OH-Leu cannot be rescued by increasing SRP concentration.

FIGURE 2.

The targeting of pPL bearing β-OH-Leu can be rescued by reducing the rate of translation elongation. A, effect of cycloheximide used in this work on the efficiency of pPL translation. B, CHX rescues the targeting defect of pPL bearing β-OH-Leu.

FIGURE 3.

Targeting of proteins with suboptimal signal sequences can be rescued by reducing translation elongation. A, CHX rescues SRP-dependent targeting of proteins with suboptimal signal sequences but has a much smaller effect on the targeting of strong SRP substrates. B, SRP exhibits a lower threshold of substrate selection in the presence of CHX.

FIGURE 4.

Mutations that compromise the kinetics of SRP-SR interaction can be rescued by reducing the rate of translation elongation. A, CHX rescues the defects of mutant FtsYs in the targeting of pPL. B, CHX rescues the defects of mutant SRP RNAs in the targeting of pPL.

FIGURE 5.

Slower translation elongation rescues the targeting defect of sub-optimal SRP substrate proteins in vivo. Failures in the efficient SRP-dependent targeting of FtsQ-PSBT (A), PhoA-Avi (B), and EspP-PSBT (C) were detected by their biotinylation in the cytoplasm, as described in the text, in wild-type (arabinose, +) and SRP-depleted (arabinose, −) cells and in the presence and absence of the translation elongation inhibitor tetracyclin.

FIGURE 6.

Protein targeting by the mammalian SRP is also subject to regulation by translation elongation rates. A, CHX rescues the targeting of proteins with suboptimal signal sequences by the mammalian SRP and SR. B, comparison of the pattern of substrate selection by the mammalian (solid line) and bacterial (dashed line) SRP/SR systems. The data for the bacterial SRP were from Fig. 3.

FIGURE 7.

Model of kinetic competition between protein synthesis and co-translational protein targeting. The ribosome-nascent chain complex can be targeted by the SRP pathway through three major steps, cargo binding (k₁ and k₋₁), SRP-SR assembly (k₂), and cargo unloading (k₃) within a limited time window, which is dictated by the rate of translation elongation (k_T) and the critical nascent chain length beyond which the SRP loses targeting competence.