The Wag31 protein interacts with AccA3 and coordinates cell wall lipid permeability and lipophilic drug resistance in Mycobacterium smegmatis

Mycobacterium tuberculosis, especially drug resistant tuberculosis, is a serious threat to global human health. Compared with other bacterial pathogens, M. tuberculosis gains stronger natural drug resistance from its unusually lipid-rich cell wall. As a DivIVA homolog, Wag31 has been demonstrated to be closely involved in peptidoglycan synthesis, cell growth and cell division. Previous research rarely investigated the role of Wag31 in drug resistance. In this study, we found Wag31 knock-down in Mycobacterium smegmatis resulted in a co-decrease of the resistance to four lipophilic drugs (rifampicin, novobiocin, erythromycin and clofazimine) and an increase in the cell permeability to lipophilic molecules. Six proteins (AccA3, AccD4 and AccD5, Fas, InhA and MmpL3) that are involved in fatty acid and mycolic acid synthesis were identified in the Wag31 interactome through Co-Immunoprecipitation. The Wag31–AccA3 interaction was confirmed by the pull-down assay. AccA3 overexpression resulted in a decrease in lipid permeability and an increase in the resistance of rifampicin and novobiocin. It confirmed the close relationship of lipophilic drug resistance, lipid permeability and the Wag31–AccA3 interaction. These results demonstrated that Wag31 maintained the resistance to lipophilic drugs and that Wag31 could play a role in controlling the lipid permeability of the cell wall through the Wag31–AccA3 interaction.     

The plant nuclear envelope protein MAF1 has an additional location at the Golgi and binds to a novel Golgi-associated coiled-coil protein

Tomato MAF1 (LeMAF1) is a plant-specific, nuclear envelope (NE)-associated protein. It is the founding member of a group of WPP domain-containing, NE-associated proteins. This group includes the Arabidopsis WPP family, which is involved in cell division, as well as plant RanGAPs. In addition to its NE localization, LeMAF1 accumulates in speckles in the cytoplasm. Here, we show that the LeMAF1-containing speckles are components of the Golgi apparatus. A novel tomato coiled-coil protein was identified that specifically binds to LeMAF1. Tomato WPP domain-associated protein (LeWAP) interacts in yeast and in vitro through its coiled-coil domain with several WPP-domain containing proteins, including AtRanGAP1 and the WPP family (LeMAF, WPP1 and WPP2). Like LeMAF1, LeWAP is localized at the Golgi. Moreover, we present data showing that Arabidopsis WAP is necessary for the existence of a multi-protein complex containing WPP2. Electronic Supplementary Material Supplementary material is available for this article at .     

Requirement of the coiled-coil domain of PML-RARalpha oncoprotein for localization, sumoylation, and inhibition of monocyte differentiation

Homo-oligomerization via a coiled-coil (C-C) domain has been shown to be necessary for the promyelocytic leukemia (PML)-retinoic acid receptor-alpha (RARalpha) fusion protein to acquire oncogenic potential in acute promyelocytic leukemia. We show here that PML(DeltaC-C)-RARalpha, which contains a deletion in its C-C domain, is neither localized as characteristic microspeckles nor modified by small ubiquitin-like modifiers (SUMO). The absence of sumoylation of the DeltaC-C mutant was due to the lack of binding to Ubc9, a SUMO conjugation enzyme. The integrity of RING finger domain was also needed for both sumoylation and microspeckle formation. In GAL4-DNA tethering assays, the DeltaC-C mutant completely lost the inhibitory effect on retinoic acid (RA)-mediated transactivation. Furthermore, the expression of CD14 in U937 cells expressing the DeltaC-C mutant in response to vitamin D3 was markedly higher than in cells expressing PML-RARalpha. However, the RA-mediated induction of C/EBPbeta in cells expressing the DeltaC-C mutant was comparable to that of control cells. Thus, our results suggest that the C-C domain-associated functions of sumoylation, localization as microspeckles, and the inhibition of monocyte differentiation all contribute to the oncogenic activity of PML-RARalpha.     

Characterization of the human GARP (Golgi associated retrograde protein) complex

The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the "orphan" SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells.     

Examination of a novel head-stalk protein family in Giardia lamblia characterised by the pairing of ankyrin repeats and coiled-coil domains

The intestinal pathogen Giardia lamblia possesses several unusual organelle features, including two equivalent nuclei, no mitochondria or peroxisomes, and a developmentally regulated rough endoplasmic reticulum and Golgi. Giardia also possesses a number of complex and unique cytoskeleton structures that dictate cell shape, motility and attachment. Our investigations of cytoskeletal proteins have revealed the presence of a new protein family. Proteins in this family contain both ankyrin repeats and coiled-coil domains; although these are common protein motifs, their pairing is unique, thus establishing a new class of head-stalk proteins. Examination of the G. lamblia genome shows evidence for at least 18 genes coding for proteins with a series of ankyrin repeats followed by a lengthy coiled-coil domain and at least an additional 14 genes coding for proteins with a prominent coiled-coil domain flanked by two series of ankyrin repeats. We have examined one of these proteins, Giardia Axoneme Associated Protein (GASP-180), in detail. GASP-180 is a 180 kDa protein containing five ankyrin repeats in a 200 amino acid N-terminal domain separated by a short spacer from an approximately 1375 amino acid coiled-coil domain. Using anti-peptide antibodies raised against a unique 20 amino acid sequence found at the C-terminus, we have determined that GASP-180 is present in cytoskeleton extractions of the parasite and localises to the proximal base of the anterior flagellar axonemes. The combination of the localisation and the structural and functional motifs of GASP-180 make it a strong candidate to participate in control of flagellar activity.     

A novel coiled-coil protein co-localizes and interacts with a calcium-dependent protein kinase in the common ice plant during low-humidity stress

McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is induced transiently by salinity and water deficit stress and also McCPK1 undergoes dynamic subcellular localization changes in response to these same stresses. Here we have confirmed that low humidity is capable of causing a drastic change in McCPK1’s subcellular localization. We attempted to elucidate this phenomenon by isolating components likely to be involved in this process. McCAP1 (M. crystallinum CDPK adapter protein 1) was cloned in a yeast two-hybrid screen with a constitutively active McCPK1 as bait. We show that McCPK1 and McCAP1 can interact in the yeast two-hybrid system, in vitro, and in vivo as demonstrated by coimmunoprecipitation experiments from plant extracts. However, McCAP1 does not appear to be a substrate for McCPK1. DsRed–McCAP1 and EGFP–McCPK1 fusions colocalize in epidermal cells of ice plants exposed to low humidity. McCAP1 is homologous to a family of proteins in Arabidopsis with no known function. Computational threading analysis suggests that McCAP1 is likely to be an intermediate filament protein of the cytoskeleton.     

Domain architecture of the atypical Arf-family GTPase Arl13b involved in cilia formation

Arl13b is an atypical Arf/Arl-family GTPase consisting of an extending large C-terminal region (C domain) and Arf-homologous GTP-binding motifs in the N terminus (N domain). Although Arl13b appears to be involved in cilia formation, its precise function and roles of the domains remain unknown. Here, we show the unique domain architecture of Arl13b by analyzing the relationship between its biochemical properties and cilia formation. Arl13b binds guanine nucleotides and specifically localizes to cilia. The ciliary localization of Arl13b requires both N and C domains but is independent of its guanine nucleotide-binding ability. Arl13b is capable of self-associating via N domain, and overexpression of N domain inhibits not only cilia formation but also the maintenance of pre-generated cilia. These findings suggest that N and C domains of Arl13b cooperatively regulate its ciliary localization and that N domain-dependent self-association of Arl13b may be important for its function in cilia biogenesis.     

A Protein Related to Eucaryal and Bacterial DNA-Motor Proteins in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius

We have isolated a new gene encoding a putative 103-kDa protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Analysis of the deduced amino-acid sequence shows an extended central domain, predicted to form coiled-coil structures, and two terminal domains that display purine NTPase motifs. These features are reminiscent of mechanochemical motor proteins which use the energy of ATP hydrolysis to move specific cellular components. Comparative analysis of the amino-acid sequence of the terminal domains and predicted structural organization of this putative purine NTPase show that it is related both to eucaryal proteins from the ``SMC family' involved in the condensation of chromosomes and to several bacterial and eucaryal proteins involved in DNA recombination/repair. Further analyses revealed that these proteins are all members of the so called ``UvrA-related NTP-binding proteins superfamily' and form a large subgroup of motor-like NTPases involved in different DNA processing mechanisms. The presence of such protein in Archaea, Bacteria, and Eucarya suggests an early origin of DNA-motor proteins that could have emerged and diversified by domain shuffling. Received: 29 June 1996 / Accepted: 28 February 1997     

BRIZ1 and BRIZ2 Proteins Form a Heteromeric E3 Ligase Complex Required for Seed Germination and Post-germination Growth in Arabidopsis thaliana

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type:growth-arrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain as follows: a BRAP2 (BRCA1-Associated Protein 2), a ZnF UBP (Zinc Finger Ubiquitin Binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form hetero-oligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and, after introduction, fail to complement their respective mutant lines. In our current model, BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.