The endoplasmic reticulum (ER) localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr518. AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr518 AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.
© 2015, Preissler et al.
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Copper is an essential enzyme cofactor in bacteria, but excess copper is highly toxic. Bacteria can cope with copper stress by increasing copper resistance and initiating chemorepellent response. However, it remains unclear how bacteria coordinate chemotaxis and resistance to copper. By screening proteins that interacted with the chemotaxis kinase CheA, we identified a copper-binding repressor CsoR that interacted with CheA in Pseudomonas putida. CsoR interacted with the HPT (P1), Dimer (P3), and HATPase_c (P4) domains of CheA and inhibited CheA autophosphorylation, resulting in decreased chemotaxis. The copper-binding of CsoR weakened its interaction with CheA, which relieved the inhibition of chemotaxis by CsoR. In addition, CsoR bound to the promoter of copper-resistance genes to inhibit gene expression, and copper-binding released CsoR from the promoter, leading to increased gene expression and copper resistance. P. putida cells exhibited a chemorepellent response to copper in a CheA-dependent manner, and CsoR inhibited the chemorepellent response to copper. Besides, the CheA-CsoR interaction also existed in proteins from several other bacterial species. Our results revealed a mechanism by which bacteria coordinately regulated chemotaxis and resistance to copper by CsoR.
5-Methylcytosine (m5C) is one of the posttranscriptional modifications in mRNA and is involved in the pathogenesis of various diseases. However, the capacity of existing assays for accurately and comprehensively transcriptome-wide m5C mapping still needs improvement. Here, we develop a detection method named DRAM (deaminase and reader protein assisted RNA methylation analysis), in which deaminases (APOBEC1 and TadA-8e) are fused with m5C reader proteins (ALYREF and YBX1) to identify the m5C sites through deamination events neighboring the methylation sites. This antibody-free and bisulfite-free approach provides transcriptome-wide editing regions which are highly overlapped with the publicly available bisulfite-sequencing (BS-seq) datasets and allows for a more stable and comprehensive identification of the m5C loci. In addition, DRAM system even supports ultralow input RNA (10 ng). We anticipate that the DRAM system could pave the way for uncovering further biological functions of m5C modifications.