The 100-kDa “a” subunit of the vacuolar proton-translocating ATPase (V-ATPase) is encoded by two genes in yeast, VPH1 and STV1. Vph1p-containing complex localizes to vacuole, whereas Stv1p-containing resides some other intracellular compartment, suggesting that a contains information necessary for correct targeting V-ATPase. We show Stv1p late Golgi compartment at steady state cycles continuously via prevacuolar endosome back Golgi. V-ATPase complexes containing Vph1p also differ their...

Ten class E Vps proteins in yeast are known components of the ESCRT complexes I, II and III, which required for sorting to lumenal membranes multivesicular bodies. We used 2 hybrid system analyze protein–protein interactions all 17 soluble proteins, as well thought be ubiquitination deubiquitination cargo at identified novel between complex suggesting that ESCRTI binds both ESCRTII ESCRTIII. These were confirmed by GST pull‐down experiments. Our data indicate link ESCRTIII is via Vps28p...

Models for protein sorting at multivesicular bodies in the endocytic pathway of mammalian cells have relied largely on data obtained from yeast. These suggest essential role four ESCRT complexes body sorting. However, putative ESCRTII complex (hVps25p, hVps22p, and hVps36p) has no proven functional endosomal transport. We characterized human investigated its function trafficking. The proteins interact with one another, hVps20p (a component ESCRTIII), their yeast homologues. Our interaction...

The last steps of multivesicular body (MVB) formation, human immunodeficiency virus (HIV)-1 budding and cytokinesis require a functional endosomal sorting complex required for transport (ESCRT) machinery to facilitate topologically equivalent membrane fission events. Increased sodium tolerance (IST) 1, new positive modulator the ESCRT pathway, has been described recently, but an essential function this highly conserved protein not identified. Here, we describe previously uncharacterized...

Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses implicated viral pathogenesis. Here we describe the cellular distribution trafficking of a human cytomegalovirus (HCMV) receptor encoded by theUS28 gene, after transient stable expression transfected HeLa Cos cells. Immunofluorescence staining indicated that this protein accumulated intracellularly vesicular structures perinuclear region cell showed overlap with markers for endocytic organelles. By...

We show that the vacuolar protein sorting gene VPS44 is identical to NHX1, a encodes sodium/proton exchanger. The Saccharomyces cerevisiae Nhx1p shows high homology mammalian exchangers of NHE family. thought transport sodium ions into prevacuole compartment in exchange for protons. Pulse-chase experiments approximately 35% newly synthesized soluble carboxypeptidase Y missorted nhx1 delta cells, and secreted from cell. cells accumulate late Golgi, prevacuole, lysosome markers an aberrant...

The Gram-negative bacterial pathogen Yersinia delivers six effector proteins into the host cells to thwart innate immune response. One of effectors, YopT, causes disruption actin cytoskeleton and contributes inhibition phagocytosis pathogen. YopT functions as a cysteine protease cleave Rho family GTPases. We have analyzed cleavage products GTPases by TLC determined their chemical structure MS. Amino acid labeling experiments were performed locate exact site in RhoA where occurs. Our data...

Cluster of differentiation antigen 4 (CD4), the T lymphocyte receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by or phorbol esters. During down-modulation CD4 dissociates from p56 lck , undergoes endocytosis through clathrin-coated pits, then sorted in early endosomes to late endocytic organelles where it degraded. Previous studies have suggested that phosphorylation a dileucine sequence required for down-modulation. Using transfected...

The ATP-gated P2X(7) receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti-inflammatory drug discovery. We have recently identified novel set of cyclic imide compounds that inhibited receptor-mediated dye uptake human macrophage THP-1 cells. In this study the actions and selectivity one these compounds, AZ11645373, were characterized.We measured membrane currents, calcium influx, YOPRO-1 from HEK cells expressing individual P2X...

A variety of techniques have been used to investigate the urea-induced kinetic folding mechanism α-subunit tryptophan synthase from Escherichia coli. distinctive property this 29 kDa α/β barrel protein is presence two stable equilibrium intermediates, populated at approximately 3 and 5 M urea. The refolding process displays multiple phases whose lifetimes span submillisecond greater than 100 s time scale; unfolding studies yield relaxation times on order 10−100 s. In an effort understand...

The Kaposi's sarcoma-associated herpes virus (KSHV) K3 viral gene product effectively down-regulates cell surface MHC class I. is an E3 ubiquitin ligase that promotes Lys(63)-linked polyubiquitination of I, providing the signal for clathrin-mediated endocytosis. Endocytosis followed by sorting into intralumenal vesicles (ILVs) multivesicular bodies (MVBs) and eventual delivery to lysosomes. I MVBs requires many individual proteins four endosomal complexes required transport (ESCRTs). In HeLa...

Despite evidence that autism is highly heritable with estimates of 15 or more genes involved, few studies have directly examined associations multiple gene interactions. Since inability to effectively combat oxidative stress has been suggested as a mechanism autism, we genetic variation 42 (308 single-nucleotide polymorphisms (SNPs)) related glutathione, the most important antioxidant in brain, for both marginal association and multi-gene interaction among 318 case-parent trios from The...

The cell surface expression of the envelope glycoproteins (Envs) primate immunodeficiency viruses is, at least in part, regulated by endocytosis signal(s) located Env cytoplasmic domain. Here, we show that a membrane proximal signal directs simian virus (SIV) to clathrin‐coated pits, and is conserved all SIV human Envs, conforms Yxxø motif (where x can be any amino acid Ø represents large hydrophobic residue). This similar described for number cellular proteins. By plasmon resonance detected...

The vacuolar-type ATPase (H + ATPase) is a ubiquitously expressed multisubunit pump whose regulation poorly understood. Its membrane-integral a-subunit involved in proton translocation and humans has four forms, a1–a4. This study investigated two naturally occurring point mutations a4's COOH terminus that cause recessive distal renal tubular acidosis (dRTA), R807Q G820R. Both lie within domain binds the glycolytic enzyme phosphofructokinase-1 (PFK-1). We recreated these disease yeast to...

The pH and lumenal environment of intracellular organelles is considered essential for protein sorting trafficking through the cell. We provide first evidence that a mammalian NHE sodium (potassium)/proton exchanger, NHE8, plays key role in control endosome morphology. At steady state, majority epitope-tagged NHE8 was found trans-Golgi network HeLa M-cells, but proportion also localized to multivesicular bodies (MVBs). Depletion M-cells with siRNA resulted perturbation MVB sorting, as shown...

Farnesylation is a posttranslational lipid modification in which 15-carbon farnesyl isoprenoid linked via thioether bond to specific cysteine residues of proteins reaction catalyzed by protein farnesyltransferase (FTase). We synthesized analogues (3−6) pyrophosphate (FPP) probe the range modifications possible FPP skeleton allow for efficient transfer FTase. Photoaffinity (5, 6) were prepared substituting perfluorophenyl azide functional groups ω-terminal isoprene FPP. Substituted anilines...

Abstract Molecular dissection was employed to identify minimal independent folding units in dihydrofolate reductase (DHFR) from Escherichia coli. Eight overlapping fragments of DHFR, spanning the entire sequence and ranging size 36 123 amino acids, were constructed by chemical cleavage. These designed examine effect tethering multiple elements secondary structure on test if structural domains represent autonomous units. CD fluorescence spectroscopy demonstrated that six containing up a total...

The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the transfer of a 15-carbon farnesyl moiety from diphosphate (FPP) to cysteine residue near C-terminus substrate. Several crystal structures inactive FTase·FPP·peptide complexes indicate that K164α interacts with α-phosphate and H248β Y300β form hydrogen bonds β-phosphate FPP [Strickland, C. L., et al. (1998) Biochemistry 37, 16601−16611]. Mutations K164Aα, H248Aβ, Y300Fβ were prepared analyzed by single turnover kinetics...

Protein farnesyltransferase (FTase) catalyzes the addition of a farnesyl chain onto sulfur C-terminal cysteine protein substrate. Magnesium ions enhance farnesylation catalyzed by FTase several hundred-fold, with <i>K</i>_Mg value 4 mm. The magnesium ion is proposed to coordinate diphosphate leaving group farnesyldiphosphate (FPP) stabilize developing charge in transition state. Here we further investigate binding site using mutagenesis and biochemical studies. Free FPP binds...

Protein farnesytransferase (FTase) catalyzes the transfer of a 15-carbon prenyl group from farnesyl diphosphate (FPP) to cysteine residue target proteins and is member newest class zinc metalloenzymes that catalyze sulfur alkylation. Common substrates FTase include oncogenic Ras proteins, therefore inhibitors are under development for treatment various cancers. An increased understanding salient features chemical transition state may aid in design potent enhance our mechanism this enzymes....