Defining a Conformational Consensus Motif in Cotransin-Sensitive Signal Sequences: A Proteomic and Site-Directed Mutagenesis Study

Abstract

The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.

A Proteomic Survey Indicates Sortilin as a Secondary Substrate of the ER Translocation Inhibitor Cyclotriazadisulfonamide (CADA)

“…It is suggested that cotransin binds to the Sec61 plug domain, near the lateral gate of the translocon channel, which prevents the inserted protein from reaching the lumen of the ER (43,44), and may explain its broad substrate range. The selectivity of cotransin was recently addressed in a proteomic study (45), which revealed that the translocation of almost all secreted proteins was affected under saturating cotransin concentrations. However, the majority of integral membrane proteins were resistant to cotransin activity.…”

Inhibitors of protein translocation across membranes of the secretory pathway: novel antimicrobial and anticancer agents

“…For example, cotransin operates selectively at low nanomolar concentrations [ 36 ]. In contrast, Klein et al have recently shown that a saturating concentration of cotransin (30 µM) actually inhibits translocation of a broad range of secreted proteins, while integral membrane proteins are mostly unaffected [ 40 ].…”

The signal peptide as a new target for drug design

“…61 On the other hand, an H-region conformational consensus motif for sensitivity of signal sequences to cotransin has been identified. 63 It was reported that specific combinations of certain amino acids were important, rather than length or hydrophobicity of the signal sequence. The nonselective translocation inhibitor apratoxin A is a macrocyclic tetrapeptide that also acts as a gating inhibitor.…”