Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

A variant of the CDKAL1 gene was reported to be associated with type 2 diabetes and reduced insulin release in humans; however, the role of CDKAL1 in β cells is largely unknown. Therefore, to determine the role of CDKAL1 in insulin release from β cells, we studied insulin release profiles in CDKAL1 gene knockout (CDKAL1 KO) mice.Total internal reflection fluorescence imaging of CDKAL1 KO β cells showed that the number of fusion events during first-phase insulin release was reduced. However, there was no significant difference in the number of fusion events during second-phase release or high K(+)-induced release between WT and KO cells. CDKAL1 deletion resulted in a delayed and slow increase in cytosolic free Ca(2+) concentration during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATP-sensitive K(+) (K(ATP)) channels to glucose was blunted in KO cells. In addition, glucose-induced ATP generation was impaired. Although CDKAL1 is homologous to cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1, there was no difference in the kinase activity of CDK5 between WT and CDKAL1 KO islets.We provide the first report describing the function of CDKAL1 in β cells. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in β cells by facilitating ATP generation, K(ATP) channel responsiveness and the subsequent activity of Ca(2+) channels through pathways other than CDK5-mediated regulation

The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome

Summary The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation and activity towards transcription or replication of the viral genome, respectively. Here, we provide evidence that the FluPol-RNAP II binding interface has a so far overlooked function for replication of the viral genome. Using a combination of cell-based and in vitro approaches, we show that the RNAP II C-terminal-domain, jointly with ANP32, enhances FluPol replication activity and we propose a model in which the host RNAP II is the anchor for transcription and replication of the viral genome. Our data open new perspectives on the spatial coupling of viral transcription and replication and the coordinated balance between these two activities

Two Fe-S clusters catalyze sulfur insertion by radical-SAM methylthiotransferases

International audienceHow living organisms create carbon-sulfur bonds during the biosynthesis of critical sulfur-containing compounds is still poorly understood. The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. Both belong to a subgroup of radical–S-adenosylmethionine (radical-SAM) enzymes that bear two [4Fe-4S] clusters. One cluster binds S-adenosylmethionine and generates an Ado• radical via a well-established mechanism. However, the precise role of the second cluster is unclear. For some sulfur-inserting radical-SAM enzymes, this cluster has been proposed to act as a sacrificial source of sulfur for the reaction. In this paper, we report parallel enzymological, spectroscopic and crystallographic investigations of RimO and MiaB, which provide what is to our knowledge the first evidence that these enzymes are true catalysts and support a new sulfation mechanism involving activation of an exogenous sulfur cosubstrate at an exchangeable coordination site on the second cluster, which remains intact during the reaction

Purification and analysis of pyrenoid preparations.

<p>(A) Pyrenoid-enriched pellet (P) fractions were obtained by solubilizing cells or purified chloroplasts (cps) with Triton X-100 followed by immediate isolation of pyrenoids by centrifugation through a Percoll cushion. Detergent-solubilized material remained in the supernatant (S). Pyrenoids and other material were recovered in the pellet (P). (B) Purification of the chloroplasts from which pyrenoid–enriched fractions were prepared is demonstrated by results of immunoblot analyses comparing extracts of cells and chloroplasts (cps) for the relative levels of marker proteins for ER (Bip), mitochondria (AOX1), cytoplasm (CyL4), and the chloroplast (L7/L12 and RbcL). Samples with 1.0 μg chlorophyll were loaded in each lane. (C and D) Results of SDS-PAGE and silver-staining reveal proteins of the P and S fractions from (C) cells and isolated chloroplasts and (D) WT and the pyrenoid-deficient control strains SSAT and <i>ΔrbcL</i>. (<i>D</i>) Asterisks indicate bands that appear to be contaminants common to P fractions from WT and at least one pyrenoid-deficient mutant. The P and S represent proportional loading of protein isolated from material containing 65 μg chlorophyll.</p

Functional annotation of the 190 proteins of the pyrenoid proteome.

<p>Proteins were classified according to KEGG for <i>C</i>. <i>reinhardtii</i> using a three Gene Ontology annotation levels. The KEGG annotations were further refined manually to optimize functional categories. Proteins involved in CO₂ fixation, CO₂ metabolism (such as carbonic anhydrases) or annotated as `low CO<sub>2</sub> inducible`genes are included in node “Photosynthesis and CO₂ metabolism”. The node “Carbohydrate” has proteins in carbohydrate (principally starch) metabolism and proteins harboring an alpha amylase catalytic domain or a starch-binding domain. Nodes are labeled either with the gene name or with the UniprotKB identifier. The functional categories and the properties of each protein are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185039#pone.0185039.s003" target="_blank">S3 Table</a>.</p

Pyrenoid-enriched fractions contain pyrenoids as revealed by IF microscopy.

<p>P fractions were shown to contain pyrenoids, seen as spherical bodies of <i>ca</i>. 1 μm that IF stain for marker proteins for the pyrenoid; RbcL and RbcS. Columns show images from differential interference contrast (DIC) microscopy, IF staining for RbcL (A-C) or RbcS (D-F), and the merged images for pyrenoid-enriched fractions from the WT strain (A and D), and the pyrenoid-deficient strains: <i>ΔrbcL</i> (B and E) and <i>SSAT</i> (C and F). Size bars = 10 μm.</p

Newly synthesized RbcL was detected in the pyrenoid fractions.

<p>Proteins in the pyrenoid-enriched P and supernatant S fractions were resolved by SDS-PAGE and analyzed by (A) silver-staining, (B) phosphorimaging of ³⁵S-labeled proteins during a 5 min pulse and (C) immunoblot analysis for RbcL. A newly synthesized ³⁵S-pulse-labeled protein with the molecular mass of RbcL (55 kDa) was detected in the pyrenoid-enriched pellet (P) fraction (RbcL). ³⁵S-pulse-labeled thylakoid membrane proteins are D1, D2 and the co-migrating α and β subunits of the ATP synthase of the chloroplast.</p