SUMMARY While cilia are recognized as important signaling organelles, the extent of ciliary functions remains unknown because of difficulties in cataloguing proteins from mammalian primary cilia. We present a method that readily captures rapid snapshots of the ciliary proteome by selectively biotinylating ciliary proteins using a cilia-targeted proximity labeling enzyme (cilia-APEX). Besides identifying known ciliary proteins, cilia-APEX uncovered several ciliary signaling molecules. The kinases PKA, AMPK and LKB1 were validated as bona fide ciliary proteins and PKA was found to regulate Hedgehog signaling in primary cilia. Furthermore, proteomics profiling of Ift27/Bbs19 mutant cilia correctly detected BBSome accumulation inside Ift27−/− cilia and revealed that β-arrestin 2 and the viral receptor CAR are candidate cargoes of the BBSome. This work demonstrates that proximity labeling can be applied to proteomics of non-membrane-enclosed organelles and suggests that proteomics profiling of cilia will enable a rapid and powerful characterization of ciliopathies.
SUMMARY Approximately one-third of global CO2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2-concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins, and physical interactors of 38 of these proteins. Our data reveal the identity of 89 pyrenoid proteins, including Rubisco-interacting proteins, photosystem I assembly factor candidates and inorganic carbon flux components. We identify three previously un-described protein layers of the pyrenoid: a plate-like layer, a mesh layer and a punctate layer. We find that the carbonic anhydrase CAH6 is in the flagella, not in the stroma that surrounds the pyrenoid as in current models. These results provide an overview of proteins operating in the eukaryotic algal CCM, a key process that drives global carbon fixation.
In solution, half-cell potentials are measured relative to those of other half cells, thereby establishing a ladder of thermochemical values that are referenced to the standard hydrogen electrode (SHE), which is arbitrarily assigned a value of exactly 0 V. Although there has been considerable interest in, and efforts toward, establishing an absolute electrochemical half-cell potential in solution, there is no general consensus regarding the best approach to obtain this value. Here, ion-electron recombination energies resulting from electron capture by gas-phase nanodrops containing individual [M(NH3)6]3+, M = Ru, Co, Os, Cr, and Ir, and Cu2+ ions are obtained from the number of water molecules that are lost from the reduced precursors. These experimental data combined with nanodrop solvation energies estimated from Born theory and solution-phase entropies estimated from limited experimental data provide absolute reduction energies for these redox couples in bulk aqueous solution. A key advantage of this approach is that solvent effects well past two solvent shells, that are difficult to model accurately, are included in these experimental measurements. By evaluating these data relative to known solution-phase reduction potentials, an absolute value for the SHE of 4.2 +/- 0.4 V versus a free electron is obtained. Although not achieved here, the uncertainty of this method could potentially be reduced to below 0.1 V, making this an attractive method for establishing an absolute electrochemical scale that bridges solution and gas-phase redox chemistry.
SUMMARY The phosphorylation of agonist-occupied G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry, electron microscopy, mutagenesis, molecular dynamics simulations and computational docking to analyze GRK5 interaction with the β2-adrenergic receptor (β2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C-terminus of the β2AR with the GRK5 RH bundle subdomain, membrane-binding surface and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs.
Hydrated divalent magnesium and calcium clusters are used as nanocalorimeters to measure the internal energy deposited into size-selected clusters upon capture of a thermally generated electron. The infrared radiation emitted from the cell and vacuum chamber surfaces as well as from the heated cathode results in some activation of these clusters, but this activation is minimal. No measurable excitation due to inelastic collisions occurs with the low-energy electrons used under these conditions. Two different dissociation pathways are observed for the divalent clusters that capture an electron: loss of water molecules (Pathway I) and loss of an H atom and water molecules (Pathway II). For Ca(H 2 O) n 2ϩ , Pathway I occurs exclusively for n Ն 30 whereas Pathway II occurs exclusively for n Յ 22 with a sharp transition in the branching ratio for these two processes that occurs for n Ϸ 24. The number of water molecules lost by both pathways increases with increasing cluster size reaching a broad maximum between n ϭ 23 and 32, and then decreases for larger clusters. From the number of water molecules that are lost from the reduced cluster, the average and maximum possible internal energy is determined to be ϳ4.4 and 5.2 eV, respectively, for Ca(. This value is approximately the same as the calculated ionization energies of M(H 2 O) n ϩ , M ϭ Mg and Ca, for large n indicating that the vast majority of the recombination energy is partitioned into internal modes of the ion and that the dissociation of these ions is statistical. For smaller clusters, estimates of the dissociation energies for the loss of H and of water molecules are obtained from theory. For Mg(H 2 O) n 2ϩ , n ϭ 4 -6, the average internal energy deposition is estimated to be 4.2-4.6 eV. The maximum possible energy deposited into the n ϭ 5 cluster is Ͻ7.1 eV, which is significantly less than the calculated recombination energy for this cluster. There does not appear to be a significant trend in the internal energy deposition with cluster size whereas the recombination energy is calculated to increase significantly for clusters with fewer than 10 water molecules. These, and other results, indicate that the dissociation of these smaller clusters is nonergodic. . The effectiveness of the bottom-up method for complex samples can be enhanced by using multidimensional separations. Clemmer and coworkers elegantly demonstrated that combining on-line liquid chromatography (LC) with ion mobility spectrometry and MS can greatly improve separations without increasing analysis times over LC/MS alone [2][3][4]. In contrast, the "top-down" approach to protein characterization has the advantage that de novo sequencing, including the identification and structural localization of labile posttranslational modifications, can be done directly on protein mixtures without proteolysis [5,6]. This top-down approach has greatly benefited from the development of electron capture dissociation (ECD), a method pioneered by McLafferty and coworkers [6][7][8][9]. In a typical ECD experim...
In solution, half-cell potentials are measured relative to other half-cells resulting in a ladder of thermodynamic values that is anchored to the standard hydrogen electrode (SHE), which is assigned an arbitrary value of exactly 0 V. A new method for measuring the absolute SHE potential is introduced in which reduction energies of Eu(H 2 O) n 3+ , from n = 55 to 140, are extrapolated as a function of the geometric dependence of the cluster reduction energy to infinite size. These measurements make it possible to directly relate absolute reduction energies of these gaseous nanodrops containing Eu 3+ to the absolute reduction enthalpy of this ion in bulk solution. From this value, an absolute SHE potential of +4.11 V and a real proton solvation energy of −269.0 kcal/mol are obtained. The infrared photodissociation spectrum of Eu(H 2 O) 119-124 3+ indicates that the structure of the surface of the nanodrops is similar to that at the bulk air-water interface and that the hydrogen bonding of interior water molecules is similar to that in aqueous solution. These results suggest that the environment of Eu 3+ in these nanodrops and the surface potential of the nandrops are comparable to those of the condensed phase. This method for obtaining absolute potentials of redox couples has the advantage that no explicit solvation model is required, which eliminates uncertainties associated with these models, making this method potentially more accurate than previous methods.
DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3′s interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5′UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3′s role in this process. Arsenite-induced stress shifts DDX3 binding from the 5′UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation.
Effects of protein conformation on electron capture dissociation (ECD) were investigated using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and Fourier-transform ion cyclotron resonance mass spectrometry. Under the conditions of these experiments, the electron capture efficiency of ubiquitin 6ϩ formed from three different solution compositions differs significantly, ranging from 51 Ϯ 7% for ions formed from an acidified water/methanol solution to 88 Ϯ 2% for ions formed from a buffered aqueous solution. This result clearly indicates that these protein ions retain a memory of their solution-phase structure and that conformational differences can be probed in an ECD experiment. Multiple conformers for the 7ϩ and 8ϩ charge states of ubiquitin were separated using FAIMS. ECD spectra of conformer selected ions of the same charge states differ both in electron capture efficiency and in the fragment ion intensities. Conformers of a given charge state that have smaller collisional cross sections can have either a larger or smaller electron capture efficiency. A greater electron capture efficiency was observed for ubiquitin 6ϩ that has the same collisional cross section as one ubiquitin 7ϩ conformer, despite the lower charge state. These results indicate that the shape of the molecule can have a greater effect on electron capture efficiency than either collisional cross section or charge state alone. The cleavage locations of different conformers of a given charge state were the same indicating that the presence of different conformers in the gas phase is not due to difference in where charges are located, but rather reflect conformational differences most likely originating from solution. Small neutral losses observed from the singly-and doubly-reduced ubiquitin 6ϩ do not show a temperature dependence to their formation, consistent with these ions being formed by nonergodic processes. [7][8][9] can be obtained. Methods based on this electron capture process appear promising for bottom up and top down approaches in proteomics due to the high information content obtained in the fragmentation spectra [10 -12]. Sequence coverage of over 90% has been reported for small proteins [13]. Several methods for implementing electron capture that use different electron sources have been reported: electron capture dissociation (ECD) with free electrons from a heated filament or cathode [1-3], electron-transfer dissociation utilizing anion reagents [14 -17], electron removal by electron impact followed by capture of a free electron in electronic excitation dissociation [18], and collisionally induced electron-transfer from neutral atoms or molecules [19,20].A consequence of the extensive fragmentation produced by ECD is that the precursor ion population can be fractionated into potentially hundreds or even thousands of fragment ions. Thus, the intensity of any particular fragment ion is typically small compared with the original precursor ion intensity [1]. High electron capture and fragmentation efficiency are cr...
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