Signal sequence-triage is activated by translocon obstruction sensed by an ER stress sensor IRE1α.

Secretory pathway proteins are cotranslationally translocated into the endoplasmic reticulum (ER) of metazoan cells through the protein channel, translocon. Given that there are far fewer translocons than ribosomes in a cell, it is essential that secretory protein-translating ribosomes only occupy translocons transiently. Therefore, if translocons are obstructed by ribosomes stalled or slowed in translational elongation, it possibly results in deleterious consequences to cellular function. Hence, we investigated how translocon clogging by stalled ribosomes affects mammalian cells. First, we constructed ER-destined translational arrest proteins (ER-TAP) as an artificial protein that clogged the translocon in the ER membrane. Here, we show that the translocon clogging by ER-TAP expression activates triage of signal sequences (SS) in which secretory pathway proteins harboring highly efficient SS are preferentially translocated into the ER lumen. Interestingly, the translocon obstructed status specifically activates inositol requiring enzyme 1α (IRE1α) but not protein kinase R-like ER kinase (PERK). Given that the IRE1α-XBP1 pathway mainly induces the translocon components, our discovery implies that lowered availability of translocon activates IRE1α, which induces translocon itself. This results in rebalance between protein influx into the ER and the cellular translocation capacity.Key words: endoplasmic reticulum, translocation capacity, translocon clogging, IRE1, signal sequence.

Influenza virus infection induces cellular Ebp1 gene expression.

Influenza virus RNA-dependent RNA polymerase is composed of three viral proteins, PB1, PB2, and PA. The host protein Ebp1 (ErbB3-binding protein1) interacts with PB1, and inhibits both in vitro RNA synthesis and virus replication. On Western blotting, the induction of Ebp1 was observed after influenza virus infection. To understand the induction of Ebp1 by influenza virus infection, we introduced a series of deletions within the 981-nucleotide long sequence located upstream of the Ebp1 gene (-664 to +317 nt from the transcription initiation site) and ligated them to the GFP gene. GFP expression assays indicated that the 981-nt upstream region was required for expression of GFP in not all cells but some cells. Microscopic analysis of the transformants showed that GFP expression was up-regulated by the influenza virus infection. Furthermore, quantitative real-time PCR indicated that influenza virus infection induced Ebp1 mRNA expression. Our data showed that (i) the newly synthesized vRNP of influenza virus induces Ebp1 expression; (ii) the Ebp1 promoter localizes between -664 nt and the initiation site of the Ebp1 gene, +317-nt long sequence in the noncoding region is required for regulation of Ebp1 gene expression; and (iii) Ebp1 expression level is correlated with virus protein expression level.