Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.     

Characterization of glycogen-synthase phosphatase and phosphorylase phosphatase in subcellular liver fractions

Upon fractionation of a postmitochondrial supernatant from rat liver, the synthase phosphatase (EC 3.1.3.42) activity (assayed at high tissue concentrations) was largely recovered in the glycogen fraction and to a minor extent in the cytosol. In contrast, the phosphorylase phosphatase (EC 3.1.3.17) activity was approximately equally distributed between these two fractions, a lesser amount being recovered in the microsomal fraction. The phosphatase activities in the microsomal and glycogen fractions were almost completely inhibited by a preincubation with the modulator protein, a specific inhibitor of type-1 (ATP,Mg-dependent) protein phosphatases. In the cytosolic fraction, however, type-2A (polycation-stimulated) phosphatase(s) contributed significantly to the dephosphorylation of phosphorylase and of in vitro phosphorylated muscular synthase. Liver synthase b, used as substrate for the measurement of synthase phosphatase throughout this work, was only activated by modulator-sensitive phosphatases. Trypsin treatment of the subcellular fractions resulted in a dramatically increased (up to 1000-fold) sensitivity to modulator, a several-fold increase in phosphorylase phosphatase activity and a complete loss of synthase phosphatase activity. Similar changes occurred during dilution of the glycogen-bound enzyme. A preincubation with the deinhibitor protein, which is known to counteract the effects of inhibitor-1 and modulator, increased several-fold the phosphorylase phosphatase activity, but exclusively in the cytosolic and microsomal fractions. It did not affect the synthase phosphatase activity. Taken together, the results indicate the existence of distinct, multi-subunit type-1 phosphatases in the cytosolic, microsomal and glycogen fractions.     

An updated assessment of the seabird populations breeding at Príncipe and Tinhosas

The Príncipe Autonomous Region is recognised as a marine biodiversity hotspot, although little is known about the status of its marine fauna. It holds most breeding seabirds of the tropical eastern Atlantic Ocean. Based on anecdotal accounts of increased fishing and seabird harvesting, regular monitoring of seabird populations is considered a priority. Therefore, a survey of Príncipe’s seabird colonies was conducted in 2017. The results revealed that the more accessible seabird colonies have disappeared. Around Príncipe, Boné de Joquei is the present main stronghold for Brown Boobies Sula leucogaster and White-tailed Tropicbirds Phaethon lepturus. The Tinhosas islands hold an estimated 300 000 seabirds, predominantly Sooty Terns Onychoprion fuscatus, but also Brown Boobies, Black Noddies Anous minutus and Brown Noddies Anous stolidus. Long-term multi-annual monitoring is needed to understand the breeding phenology of each species and to better assess population trends. Ensuring a protective status for both Tinhosas and the seabirds under national legislation is a key priority for future conservation policy in São Tomé and Príncipe.     

Protein phosphatase-1 is a novel regulator of the interaction between IRBIT and the inositol 1,4,5-trisphosphate receptor

IRBIT is an IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP3 for binding to the IP3R. Phosphorylation of IRBIT is essential for the interaction with the IP3R. The unique N-terminal region of IRBIT, residues 1-104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser68 of IRBIT. Phosphorylation of Ser68 was required for subsequent phosphorylation of Ser71 and Ser74, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser71 and Ser74 were sufficient to enable inhibition of IP3 binding to the IP3R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP3R. This pinpoints PP1 as a key player in the regulation of IP3R-controlled Ca2+ signals.     

A complex of catalytically inactive protein phosphatase-1 sandwiched between Sds22 and inhibitor-3

Protein Ser/Thr phosphatase-1 (PP1) associates with a host of proteins to form substrate-specific holoenzymes. Sds22 and Inhibitor-3 (I3) are two independently described ancient interactors of PP1. We show here by various approaches that Sds22 and I3 form a heterotrimeric complex with PP1, both in cell lysates and after purification. The stability of the complex depended on functional PP1 interaction sites in Sds22 and I3, indicating that PP1 is sandwiched between Sds22 and I3. Intriguingly, I3 could not be replaced in this complex by another PP1 interactor with the same PP1 binding motif. In vitro, Sds22 and I3 were potent inhibitors of PP1, but with only some substrates. The inhibition by Sds22 could be reproduced with synthetic Sds22 fragments comprising leucine-rich repeats (LRR) 2 and 5. Sds22 and LRR5 also slowly converted PP1 into a conformation that was inactive with all tested substrates. Cell lysates that were prepared under conditions that prevented the Sds22-induced inactivation of PP1 contained a catalytically inactive complex of Sds22, PP1, and I3, indicating that this complex exists in vivo. Therefore, our studies show that a pool of PP1 is complexly controlled by both Sds22 and I3.     

How to quantify protein diffusion in the bacterial membrane

Lateral diffusion of proteins in the plane of a biological membrane is important for many vital processes, including energy conversion, signaling, chemotaxis, cell division, protein insertion, and secretion. In bacteria, all these functions are located in a single membrane. Therefore, quantitative measurements of protein diffusion in bacterial membranes can provide insight into many important processes. Diffusion of membrane proteins in eukaryotes has been studied in detail using various experimental techniques, including fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and particle tracking using single-molecule fluorescence (SMF) microscopy. In case of bacteria, such experiments are intrinsically difficult due to the small size of the cells. Here, we review these experimental approaches to quantify diffusion in general and their strengths and weaknesses when applied to bacteria. In addition, we propose a method to extract multiple diffusion coefficients from trajectories obtained from SMF data, using cumulative probability distributions (CPDs). We demonstrate the power of this approach by quantifying the heterogeneous diffusion of the bacterial membrane protein TatA, which forms a pore for the translocation of folded proteins. Using computer simulations, we study the effect of cell dimensions and membrane curvature on measured CPDs. We find that at least two mobile populations with distinct diffusion coefficients (of 7 and 169 nm(2) ms(-1) , respectively) are necessary to explain the experimental data. The approach described here should be widely applicable for the quantification of membrane-protein diffusion in living bacteria.