Overlaps between the various biodegradation pathways in Sphingomonas subarctica SA1

A bacterium capable to grow on sulfanilic acid as sole carbon, nitrogen and sulfur source has been isolated. A unique feature of this strain is that it contains the full set of enzymes necessary for the biodegradation of sulfanilic acid. Taxonomical analysis identified our isolate as Sphingomonas subarctica SA1 sp. The biodegradation pathway of sulfanilic acid was investigated at the molecular level. Screening the substrate specificity of the strain disclosed its capacity to degrade six analogous aromatic compounds including p -aminobenzoic acid. Moreover, the strain was successfully used for removal of oil contaminations. S. subarctica SA1 seemed to use distinct enzyme cascades for decomposition of these molecules, since alternative enzymes were induced in cells grown on various substrates. However, the protein patterns appearing upon induction by sulfanilic acid and sulfocatechol were very similar to each other indicating common pathways for the degradation of these substrates. Cells grown on sulfanilic acid could convert p -aminobenzoic acid to some extent and vice versa. Two types of ring cleaving dioxygenases were detected in the cells grown on various substrates: one preferred protocatechol, while the other had higher activity with sulfocatechol. This latter enzyme, named as sulfocatechol dioxygenase was partially purified and characterized

DYNLT (Tctex-1) forms a tripartite complex with dynein intermediate chain and RagA, hence linking this small GTPase to the dynein motor

© 2015 FEBS. It has been suggested that DYNLT, a dynein light chain known to bind to various cellular and viral proteins, can function as a microtubule-cargo adaptor. Recent data showed that DYNLT links the small GTPase Rab3D to microtubules and, for this to occur, the DYNLT homodimer needs to display a binding site for dynein intermediate chain together with a binding site for the small GTPase. We have analysed in detail how RagA, another small GTPase, associates to DYNLT. After narrowing down the binding site of RagA to DYNLT we could identify that a β strand, part of the RagA G3 box involved in nucleotide binding, mediates this association. Interestingly, we show that both microtubule-associated DYNLT and cytoplasmic DYNLT are equally able to bind to the small GTPases Rab3D and RagA. Using NMR spectroscopy, we analysed the binding of dynein intermediate chain and RagA to mammalian DYNLT. Our experiments identify residues of DYNLT affected by dynein intermediate chain binding and residues affected by RagA binding, hence distinguishing the docking site for each of them. In summary, our results shed light on the mechanisms adopted by DYNLT when binding to protein cargoes that become transported alongside microtubules bound to the dynein motor.Peer Reviewe

LC8 dynein light chain (DYNLL1) binds to the C-terminal domain of ATM-interacting protein (ATMIN/ASCIZ) and regulates its subcellular localization

LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1. Thus, our results imply a potential cellular interference between DYNLL1 and ATMIN functions.This work was supported by Grants MICINN BFU2009-10442 (I.R.C.), Consolider-centrosoma 3D (CSD2006-00023) (M.B.), CTQ2008-0080 (M.B.), OTKA NK81950 (L.N.), K81934 (K.S.), TÁMOP 4.2.1./B-09/KMR-2010-0003

Structure of the dynein-2 complex and its assembly with intraflagellar transport trains

Dynein-2 assembles with polymeric intraflagellar transport (IFT) trains to form a transport machinery crucial for cilia biogenesis and signaling. Here we recombinantly expressed the ~1.4 MDa human dynein-2 complex and solved its cryo-EM structure to near-atomic resolution. The two identical copies of the dynein-2 heavy chain are contorted into different conformations by a WDR60-WDR34 heterodimer and a block of two RB and six LC8 light chains. One heavy chain is steered into a zig-zag, which matches the periodicity of the anterograde IFT-B train. Contacts between adjacent dyneins along the train indicate a cooperative mode of assembly. Removal of the WDR60-WDR34-light chain subcomplex renders dynein-2 monomeric and relieves auto-inhibition of its motility. Our results converge on a model in which an unusual stoichiometry of non-motor subunits control dynein-2 assembly, asymmetry, and activity, giving mechanistic insight into dynein-2’s interaction with IFT trains and the origin of diverse functions in the dynein family

The yeast dynein Dyn2-Pac11 complex is a dynein dimerization/processivity factor: structural and single-molecule characterization

Cytoplasmic dynein is the major microtubule minus end–directed motor. Although studies have probed the mechanism of the C-terminal motor domain, if and how dynein's N-terminal tail and the accessory chains it binds regulate motor activity remain to be determined. Here, we investigate the structure and function of the Saccharomyces cerevisiae dynein light (Dyn2) and intermediate (Pac11) chains in dynein heavy chain (Dyn1) movement. We present the crystal structure of a Dyn2-Pac11 complex, showing Dyn2-mediated Pac11 dimerization. To determine the molecular effects of Dyn2 and Pac11 on Dyn1 function, we generated dyn2Δ and dyn2Δpac11Δ strains and analyzed Dyn1 single-molecule motor activity. We find that the Dyn2-Pac11 complex promotes Dyn1 homodimerization and potentiates processivity. The absence of Dyn2 and Pac11 yields motors with decreased velocity, dramatically reduced processivity, increased monomerization, aggregation, and immobility as determined by single-molecule measurements. Deleting dyn2 significantly reduces Pac11-Dyn1 complex formation, yielding Dyn1 motors with activity similar to Dyn1 from the dyn2Δpac11Δ strain. Of interest, motor phenotypes resulting from Dyn2-Pac11 complex depletion bear similarity to a point mutation in the mammalian dynein N-terminal tail (Loa), highlighting this region as a conserved, regulatory motor element