CoESPRIT: A Library-Based Construct Screening Method for Identification and Expression of Soluble Protein Complexes

Structural and biophysical studies of protein complexes require multi-milligram quantities of soluble material. Subunits are often unstable when expressed separately so co-expression strategies are commonly employed since in vivo complex formation can provide stabilising effects. Defining constructs for subunit co-expression experiments is difficult if the proteins are poorly understood. Even more problematic is when subunit polypeptide chains co-fold since individually they do not have predictable domains. We have developed CoESPRIT, a modified version of the ESPRIT random library construct screen used previously on single proteins, to express soluble protein complexes. A random library of target constructs is screened against a fixed bait protein to identify stable complexes. In a proof-of-principle study, C-terminal fragments of the influenza polymerase PB2 subunit containing folded domains were isolated using importin alpha as bait. Separately, a C-terminal fragment of the PB1 subunit was used as bait to trap N-terminal fragments of PB2 resulting in co-folded complexes. Subsequent expression of the target protein without the bait indicates whether the target is independently stable, or co-folds with its partner. This highly automated method provides an efficient strategy for obtaining recombinant protein complexes at yields compatible with structural, biophysical and functional studies

The Glyceraldehyde-3-Phosphate Dehydrogenase and the Small GTPase Rab 2 Are Crucial for Brucella Replication

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the “Brucella-containing vacuole” (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC ι, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells

Gale crater and impact processes – Curiosity’s first 364 Sols on Mars

Impact processes at all scales have been involved in the formation and subsequent evolution of Gale crater. Small impact craters in the vicinity of the Curiosity MSL landing site and rover traverse during the 364 Sols after landing have been studied both from orbit and the surface. Evidence for the effect of impacts on basement outcrops may include loose blocks of sandstone and conglomerate, and disrupted (fractured) sedimentary layers, which are not obviously displaced by erosion. Impact ejecta blankets are likely to be present, but in the absence of distinct glass or impact melt phases are difficult to distinguish from sedimentary/volcaniclastic breccia and conglomerate deposits. The occurrence of individual blocks with diverse petrological characteristics, including igneous textures, have been identified across the surface of Bradbury Rise, and some of these blocks may represent distal ejecta from larger craters in the vicinity of Gale. Distal ejecta may also occur in the form of impact spherules identified in the sediments and drift material. Possible examples of impactites in the form of shatter cones, shocked rocks, and ropy textured fragments of materials that may have been molten have been observed, but cannot be uniquely confirmed. Modification by aeolian processes of craters smaller than 40 m in diameter observed in this study, are indicated by erosion of crater rims, and infill of craters with aeolian and airfall dust deposits. Estimates for resurfacing suggest that craters less than 15 m in diameter may represent steady state between production and destruction. The smallest candidate impact crater observed is ~0.6 m in diameter. The observed crater record and other data are consistent with a resurfacing rate of the order of 10 mm/Myr; considerably greater than the rate from impact cratering alone, but remarkably lower than terrestrial erosion rates

Ubiquitin-dependent lysosomal targeting of GABAA receptors regulates neuronal inhibition

The strength of synaptic inhibition depends partly on the number of GABAA receptors (GABAARs) found at synaptic sites. The trafficking of GABAARs within the endocytic pathway is a key determinant of surface GABAAR number and is altered in neuropathologies, such as cerebral ischemia. However, the molecular mechanisms and signaling pathways that regulate this trafficking are poorly understood. Here, we report the subunit specific lysosomal targeting of synaptic GABAARs. We demonstrate that the targeting of synaptic GABAARs into the degradation pathway is facilitated by ubiquitination of a motif within the intracellular domain of the γ2 subunit. Blockade of lysosomal activity or disruption of the trafficking of ubiquitinated cargo to lysosomes specifically increases the efficacy of synaptic inhibition without altering excitatory currents. Moreover, mutation of the ubiquitination site within the γ2 subunit retards the lysosomal targeting of GABAARs and is sufficient to block the loss of synaptic GABAARs after anoxic insult. Together, our results establish a previously unknown mechanism for influencing inhibitory transmission under normal and pathological conditions