Structure and function of the bacterial heterodimeric ABC transporter CydDC: stimulation of ATPase activity by thiol and heme compounds.

In Escherichia coli, the biogenesis of both cytochrome bd-type quinol oxidases and periplasmic cytochromes requires the ATP-binding cassette-type cysteine/GSH transporter, CydDC. Recombinant CydDC was purified as a heterodimer and found to be an active ATPase both in soluble form with detergent and when reconstituted into a lipid environment. Two-dimensional crystals of CydDC were analyzed by electron cryomicroscopy, and the protein was shown to be made up of two non-identical domains corresponding to the putative CydD and CydC subunits, with dimensions characteristic of other ATP-binding cassette transporters. CydDC binds heme b. Detergent-solubilized CydDC appears to adopt at least two structural states, each associated with a characteristic level of bound heme. The purified protein in detergent showed a weak basal ATPase activity (approximately 100 nmol Pi/min/mg) that was stimulated ∼3-fold by various thiol compounds, suggesting that CydDC could act as a thiol transporter. The presence of heme (either intrinsic or added in the form of hemin) led to a further enhancement of thiol-stimulated ATPase activity, although a large excess of heme inhibited activity. Similar responses of the ATPase activity were observed with CydDC reconstituted into E. coli lipids. These results suggest that heme may have a regulatory role in CydDC-mediated transmembrane thiol transport

Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis

During endocytosis in S. cerevisiae, actin polymerization is proposed to provide the driving force for invagination against the effects of turgor pressure. In previous studies, Ysc84 was demonstrated to bind actin through a conserved N-terminal domain. However, full length Ysc84 could only bind actin when its C-terminal SH3 domain also bound to the yeast WASP homologue Las17. Live cell-imaging has revealed that Ysc84 localizes to endocytic sites after Las17/WASP but before other known actin binding proteins, suggesting it is likely to function at an early stage of membrane invagination. While there are homologues of Ysc84 in other organisms, including its human homologue SH3yl-1, little is known of its mode of interaction with actin or how this interaction affects actin filament dynamics. Here we identify key residues involved both in Ysc84 actin and lipid binding, and demonstrate that its actin binding activity is negatively regulated by PI(4,5)P2. Ysc84 mutants defective in their lipid or actin-binding interaction were characterized in vivo. The abilities of Ysc84 to bind Las17 through its C-terminal SH3 domain, or to actin and lipid through the N-terminal domain were all shown to be essential in order to rescue temperature sensitive growth in a strain requiring YSC84 expression. Live cell imaging in strains with fluorescently tagged endocytic reporter proteins revealed distinct phenotypes for the mutants indicating the importance of these interactions for regulating key stages of endocytosis

26th Annual Computational Neuroscience Meeting (CNS*2017): Part 3 - Meeting Abstracts - Antwerp, Belgium. 15–20 July 2017

This work was produced as part of the activities of FAPESP Research,\ud Disseminations and Innovation Center for Neuromathematics (grant\ud 2013/07699-0, S. Paulo Research Foundation). NLK is supported by a\ud FAPESP postdoctoral fellowship (grant 2016/03855-5). ACR is partially\ud supported by a CNPq fellowship (grant 306251/2014-0)

Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis

Dynamins are a conserved family of proteins involved in many membrane fusion and fission events. Previously, the dynamin-related protein Vps1 was shown to localize to endocytic sites, and yeast carrying deletions for genes encoding both the BAR domain protein Rvs167 and Vps1 had a more severe endocytic scission defect than either deletion alone. Vps1 and Rvs167 localize to endocytic sites at the onset of invagination and disassemble concomitant with inward vesicle movement. Rvs167-GFP localization is reduced in cells lacking vps1 suggesting that Vps1 influences Rvs167 association with the endocytic complex. Unlike classical dynamins, Vps1 does not have a proline–arginine domain that could interact with SH3 domain-containing proteins. Thus, while Rvs167 has an SH3 domain, it is not clear how an interaction would be mediated. Here, we demonstrate an interaction between Rvs167 SH3 domain and the single type ISH3-binding motif in Vps1. Mutant Vps1 that cannot bind Rvs167 rescues all membrane fusion/fission functions associated with Vps1 except for endocytic function, demonstrating the specificity and mechanistic importance of the interaction. In vitro, an Rvs161/Rvs167 heterodimer can disassemble Vps1 oligomers. Overall, the data support the idea that Vps1 and the amphiphysins function together to mediate scission during endocytosis in yeast

Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1

The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process

Ysc84 overexpression affects lifetime of different endocytic markers.

<p>(A) The effect of Ysc84 overexpression on endocytosis was assessed by measuring lifetime of different endocytic reporters. Movies of cells expressing fluorescently tagged different endocytic markers transformed with <i>YSC84</i> and an empty plasmid were recorded. At least 30 patches were assessed for each transformant. Error bars are SD. The differences in lifetimes of endocytic markers were calculated using two-tailed Student’s <i>t</i>-test. *** indicates a P value of 0.002 and **** ≥ 0.001. (B) Kymographs were generated from individual patches of fluorescently tagged endocytic markers expressing an empty plasmid or overexpressing Y<i>SC84</i> using the multiple kymographs ImageJ plugin. Arrows mark retractions. (C) Spot tracking of Sla1-GFP patches was performed in cells expressing an empty plasmid or overexpressing <i>YSC84</i> in manual tracking–ImageJ. Red spot–start, green spot–end. The time between spots is 1 second.</p

Ysc84 and Rvs167 bind to a common site on Las17.

<p>(A) Yeast two-hybrid analysis was carried out in which the polyproline domain of Las17 was carried on an activation plasmid and the Ysc84 or Rvs167 SH3 domains were expressed on the Gal4 binding domain (bait) plasmid. Mutations of Las17 carrying P387A and P388A mutations were used to reveal inhibition of binding of both SH3 domains. (B) Membrane dot blots carrying overlapping 12mer peptides of Las17 over the region 373–406 were incubated with GST fused SH3 domains of Ysc84 and Rvs167 or with GST alone. Proteins were detected by western blotting for GST.</p

Rescue of actin and growth phenotypes of ysc84∆, lsb5(1–142) strain.

<p><i>ysc84∆</i>, <i>lsb5(1–142)</i> strain was transformed with plasmids carrying wild-type or mutant <i>ysc84</i> or with an empty plasmid. (A) Growth on plates was assessed at 30°C and 37°C. (B) Cells were fixed and stained with rhodamine phalloidin before analysing actin organization microscopically. Actin organization in the cells was categorized as detailed.</p

Lipid binding activity of Ysc84-Nt.

<p>(A) Dot-blot assays were performed on PIP strips to determine the specificity of Ysc84 wild-type and mutants against 15 phospholipids. The membrane was incubated with 10 μM of His-tagged purified protein and proteins detected using anti-His tag antibodies. (B) Liposome co-sedimentation assay was performed using Ysc84-Nt and 70% PE, 30% PC-based liposomes supplemented in right lanes with 10% of PI(4,5)P₂. Proteins in the supernatant (S) and pellet (P) were visualized by Coomassie staining. Densitometry was used to determine the proportion of protein pelleting with liposomes (lower panel). Results are mean (±SD) of two independent experiments. Kinetics of F-actin barbed end elongation in the absence and presence of Ysc84 and PI(4,5)P₂. (C,D) In a pyrene-based fluorimetry assay pre-formed actin seeds (1 μM) were mixed with 1 μM G-actin and incubated with wild type and KK (C) or wild type and LK (D) Ysc84-Nt in the absence and presence of 1.2 μM PI(4,5)P₂.</p