Intracellular cycling of lysosomal enzyme receptors: cytoplasmic tails' tales.
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ADP-Ribosylation Factors↗
Biomedical subjects
G S Payne
Publications and source records attributed to G S Payne.
Radio-frequency probe for 1H decoupled 31P MRS of the head and neck region.
For optimal performance of 31P MRS at 1.5 Tesla, the use of a double resonant probe is essential to enable the application of 1H decoupling and Nuclear Overhauser Enhancement. This note describes the design, evaluation and safety validation of a versatile and compact probe optimized for 1H decoupled 31P MRS studies of tumors close to the surface of the body, in particular the head and neck region.
Equipment Design↗
Clathrin interactions with C-terminal regions of the yeast AP-1 beta and gamma subunits are important for AP-1 association with clathrin coats.
Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo recruitment and clathrin assembly during clathrin-coated vesicle biogenesis. We have identified, and characterized the physiological significance of clathrin-binding activities in the two large subunits of the AP-1 complex in Saccharomyces cerevisiae. Using GST-fusion chromatography, two clathrin-binding sites were defined in the beta1 subunit that match consensus clathrin-binding sequences in other mammalian and yeast clathrin-binding proteins. Clathrin interactions were also identified with the C-terminal region of the gamma subunit. When introduced into chromosomal genes, point mutations in the beta1 clathrin-binding motifs, or deletion of the gamma C-terminal region, reduced association of AP-1 with clathrin in coimmunoprecipitation assays. The beta1 mutations or the gamma truncation individually produced minor effects on AP-1 distribution by subcellular fractionation. However, when beta1 and gamma mutations were combined, severe defects were observed in AP-1 association with membranes and incorporation into clathrin-coated vesicles. The combination of subunit mutations accentuated growth and alpha-factor pheromone maturation defects in chc1-ts cells, though not to the extent caused by complete loss of AP-1 activity. Our results suggest that both the beta1 and gamma subunits contribute interactions with clathrin that are important for stable assembly of AP-1 complexes into clathrin coats in vivo.
Adaptor Protein Complex beta Subunits↗
Ric1p and the Ypt6p GTPase function in a common pathway required for localization of trans-Golgi network membrane proteins.
In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1Delta) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1Delta cells also harboring end3Delta to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes. SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1Delta. Further comparison of ric1Delta and ypt6Delta cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t-SNAREs, partially suppressed phenotypes of ric1Delta and ypt6Delta cells. SLY1-20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi network.
Fungal Proteins↗
Vps10p transport from the trans-Golgi network to the endosome is mediated by clathrin-coated vesicles.
A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.
Adaptor Protein Complex beta Subunits↗
Yeast Gga coat proteins function with clathrin in Golgi to endosome transport.
Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the "ear" domain of the clathrin adaptor AP-1 gamma subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2Delta), the major Gga protein, accentuates growth and alpha-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2Delta or a deletion of the AP-1 beta subunit gene (apl2Delta) alone are phenotypically normal, but cells carrying both gga2Delta and apl2Delta are defective in growth, alpha-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.
ADP-Ribosylation Factors↗
Effects of chronic alcohol consumption on the broad phospholipid signal in human brain: an in vivo 31P MRS study.
BACKGROUND: Phosphorus magnetic resonance spectroscopy (31P MRS) allows for the measurement of phospholipids and their breakdown products in the human brain. Fairly mobile membrane phospholipids give rise to a broad signal that co-resonates with metabolic phosphodiesters. Chronic alcohol exposure increases the rigidity of isolated brain membranes and, thus, may affect the amount and transverse relaxation times (T2) of MRS-detectable phospholipids. We tested the hypothesis that subjects who were heavy drinkers have stiffer membranes than controls who were light drinkers, as reflected in a smaller broad signal component and a shorter T2 of the broad signal in 31P MR spectra of the brain. METHODS: Thirteen alcohol-dependent heavy drinkers (mean age 44 years) were studied by localized 31P MRS in the centrum semiovale and compared with 17 nondependent light drinkers of similar age. The broad component signal was separated from the metabolite signal by convolution difference, which is based on the large difference in line widths of these two signals. Longitudinal and T2 relaxation times were measured using standard methods. RESULTS: The broad component integral was 13% lower in the brain of heavy drinkers compared with light drinkers (p < 0.001) and remained significantly smaller after corrections for both longitudinal and transverse relaxations (p < 0.01). The T2 distribution of the broad component consistently showed two resolvable components in both groups. The fast relaxing component had the same T2 in both groups (T2 = 1.9 msec). The slower relaxing component T2 was 0.6 msec shorter in heavy drinkers compared with light drinkers (p = 0.08). CONCLUSIONS: These results, observed in the absence of white matter volume loss, are consistent with biochemical alterations and higher rigidity of white matter phospholipids associated with long-term chronic alcohol abuse. The observed smaller broad signal component in these relatively young heavy drinkers is a sensitive measure of white matter phospholipid damage.
Adult↗
Effects of abstinence from alcohol on the broad phospholipid signal in human brain: an in vivo 31P magnetic resonance spectroscopy study.
BACKGROUND: In vivo phosphorus magnetic resonance spectroscopy (31P MRS) at a magnetic field strength of 1.5 T allows measurement of fairly mobile membrane phospholipids in the human brain. We previously showed that subjects who are heavy drinkers had a smaller signal and a shorter transverse relaxation time (T2) of white matter phospholipids than light drinkers, which suggested lower concentrations and molecular mobility of phospholipids in heavy drinkers. The purpose of the present study was to measure if such chronic alcohol-induced white matter tissue changes are persistent in long-term abstinent alcoholics. METHODS: Fourteen abstinent alcoholics (mean age 45 years, seven men and seven women) were studied by localized 31P MRS in the centrum semiovale and were compared with 13 male, alcohol-dependent, heavy drinkers and 23 nondependent light drinkers (17 men, 6 women) of similar age. Methods for measurements of the broad membrane phospholipid signal and its relaxation time were described previously. RESULTS: Phospholipid concentrations and relaxation times in alcoholics abstinent for an average of 31 months were not significantly different from those measured in light drinkers. The contribution of fast and slowly relaxing signal components to the broad phospholipid signal, however, was still different in abstinent alcoholics compared with light drinkers. No effects of sex or of family history of alcoholism were noted on any of our spectroscopic measures within the light-drinking or abstinent groups. CONCLUSIONS: Most of our results suggest at least partial recovery of chronic alcohol-induced white matter phospholipid damage with long-term abstinence. They offer myelination changes and/or dendritic rearborization as a possible mechanism for the commonly observed white matter volume gain with prolonged abstinence. But the results also suggest a persistent abnormality in the nature and/or physical properties of white matter phospholipids in long-term abstinent alcoholics.
Adult↗
Surface-coil polarization transfer for monitoring tissue metabolism in vivo.
Polarization transfer methods can substantially enhance NMR signals from nuclei of low gamma, which are J-coupled to nuclei of high gamma, but to date have had limited application for in vivo (31)P MRS studies. They require both accurate flip angles and good localization in order to achieve their potential. Described here is an implementation of the insensitive nuclei enhanced by polarization transfer (INEPT) polarization transfer method using segmented adiabatic BIR4 RF pulses. Localization uses ISIS, applied to the coupled (1)H spins. Detailed analysis is performed to evaluate the specific absorption rate power deposition when using surface coils. Polarization transfer, localization capability, and use with surface coils are demonstrated using suitable test objects. Finally, in vivo data are presented from the liver of a normal volunteer in which the signals from the phosphodiester peaks are substantially enhanced.
Cell Polarity↗
Intravenous delivery of hyperpolarized (129)Xe: a compartmental model.
There is an increasing interest in the use of hyperpolarized 129-xenon (HpXe) NMR for the measurement of tissue perfusion. In this paper we present a theoretical study designed to assess the merit of intravenous HpXe delivery compared with the existing respiration techniques. A compartmental model was created to describe the behavior of the injected bolus in the circulatory system and in the lungs. The dependence of the tissue concentration on the T(1) and solubility of the Xe in the various compartments, and on injection rate, were evaluated. By this process the critical loss mechanisms are identified. It is shown that the predicted tissue concentrations of HpXe in gray and white matter are comparable using respiration or injection techniques.
Humans↗
1H decoupling for in vivo (19)F MRS studies using the time-share modulation method on a clinical 1.5 T NMR system.
Time-share modulation has been implemented successfully on a clinical 1.5 T nuclear magnetic resonance (NMR) system to perform in vivo (19)F[-(1)H] decoupling. It uses commercially available radiofrequency (RF) filters, a transistor-transistor logic (TTL)-controlled attenuator, and a double-resonant RF surface coil suitable for (19)F magnetic resonance spectroscopy (MRS) studies in vivo. This decoupling scheme gives a threefold improvement in signal-to-noise ratio performance compared with the conventional WALTZ-4 decoupling scheme, when significant interaction exists between the decoupler and the receiver. It can also eliminate receiver saturation (i.e., saturation due to coupling of the strong proton decoupling pulse to the (19)F coil) without the need for a high-isolation coil system, thus allowing flexibility in coil geometry. This method has been demonstrated in vivo in patients receiving 5-fluorouracil chemotherapy. Magn Reson Med 44:5-9, 2000.
Antimetabolites, Antineoplastic↗
Initial measurements of ifosfamide and cyclophosphamide in patients using (31)P MRS: pulse-and-acquire, decoupling, and polarization transfer.
Ifosfamide and cyclophosphamide are (31)P-containing alkylating agents used widely in the treatment of cancer. In this communication it is demonstrated that signals from these agents may be detected in the livers of patients undergoing treatment using (31)P MRS at 1.5 T. In vitro, signals are enhanced 4-fold by use of (1)H-decoupling, with a B(1) field of 100 Hz at -150 Hz relative to water. Polarization transfer (BINEPT) enhances signals in vitro by a further factor of 5.5. Preliminary results using the double-resonance methods in vivo show that the technique is practicable although enhancements may be less than observed in vitro. Factors affecting signal enhancement in vivo are evaluated. Magn Reson Med 44:180-184, 2000.
Antineoplastic Agents, Alkylating↗
SAR and tissue heating with a clinical (31)P MRS protocol using surface coils, adiabatic pulses, and proton-decoupling.
In MRS studies using surface transmit coils, accurate assessment of local SAR and RF heating represents a difficult problem involving the coil geometry and electromagnetic and geometric tissue properties. Methodologies to determine the optimum operating parameters for dual-resonant surface coil measurements are presented, based on a standardized coil and protocol used in a multicenter (31)P MRS clinical trial, using adiabatic pulses and bilevel proton decoupling. Spatial distributions of absorbed radiation in human calf and in a tissue-equivalent gel phantom were modeled using finite-element simulations and realistic conductivity and permittivity values. Local SAR in worst-case 1 cm(3) volumes of interest (VOIs) in calf is predicted to be below international guidelines, and the temperature at the skin surface was found to increase due to the RF by less than 2 degrees C and remain below 37 degrees C. The heating rate and maximum temperature in the gel, at positions guided by the simulations, were within guideline values for both extremities and trunk and in reasonable agreement with that predicted.
Adult↗
A yeast DNA J protein required for uncoating of clathrin-coated vesicles in vivo.
Clathrin-coated vesicles mediate diverse processes such as nutrient uptake, downregulation of hormone receptors, formation of synaptic vesicles, virus entry, and transport of biosynthetic proteins to lysosomes. Cycles of coat assembly and disassembly are integral features of clathrin-mediated vesicular transport (Fig. 1a). Coat assembly involves recruitment of clathrin triskelia, adaptor complexes and other factors that influence coat assembly, cargo sequestration, membrane invagination and scission (Fig. 1a). Coat disassembly is thought to be essential for fusion of vesicles with target membranes and for recycling components of clathrin coats to the cytoplasm for further rounds of vesicle formation. In vitro, cytosolic heat-shock protein 70 (Hsp70) and the J-domain co-chaperone auxilin catalyse coat disassembly. However, a specific function of these factors in uncoating in vivo has not been demonstrated, leaving the physiological mechanism and significance of uncoating unclear. Here we report the identification and characterization of a Saccharomyces cerevisiae J-domain protein, Aux1. Inactivation of Aux1 results in accumulation of clathrin-coated vesicles, impaired cargo delivery, and an increased ratio of vesicle-associated to cytoplasmic clathrin. Our results demonstrate an in vivo uncoating function of a J domain co-chaperone and establish the physiological significance of uncoating in transport mediated by clathrin-coated vesicles.
Adaptor Proteins, Vesicular Transport↗
Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network.
Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane. To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae. Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone alpha-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment. By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN. One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences. The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1. Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53, but not the related INP51 and INP52 genes, resulted in alpha-factor maturation defects and exacerbated alpha-factor maturation defects when combined with chc1-521. Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN.
Base Sequence↗
Magnetic resonance spectroscopy in the evaluation of neurotoxicity following cranial irradiation for childhood cancer.
In order to evaluate the role of proton MR spectroscopy (1H-MRS) in the diagnosis and assessment of long-term radiation-related neurotoxicity, 14 children who had received cranial irradiation for the treatment of childhood leukaemia (n = 6) or brain tumours (n = 8) underwent 1H-MRS, MRI and neuropsychological assessment. Short-term effects at 2 months following treatment were studied in a further three patients. MRI abnormalities were observed in nine patients. No statistically significant differences between patients and controls (n = 17) were seen in any of the calculated 1H-MRS metabolite ratios, in any of the three patient groups. On multivariate logistic regression analysis there was a correlation between the choline/water ratio and a low IQ. It is concluded that any systematic radiation-induced changes in the 1H MRS metabolites must be below the detection threshold of this study.
Adolescent↗
Adaptor complex-independent clathrin function in yeast.
Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and alpha-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or alpha-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the beta subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.
Adaptor Proteins, Vesicular Transport↗
A modulatory role for clathrin light chain phosphorylation in Golgi membrane protein localization during vegetative growth and during the mating response of Saccharomyces cerevisiae.
The role of clathrin light chain phosphorylation in regulating clathrin function has been examined in Saccharomyces cerevisiae. The phosphorylation state of yeast clathrin light chain (Clc1p) in vivo was monitored by [32P]phosphate labeling and immunoprecipitation. Clc1p was phosphorylated in growing cells and also hyperphosphorylated upon activation of the mating response signal transduction pathway. Mating pheromone-stimulated hyperphosphorylation of Clc1p was dependent on the mating response signal transduction pathway MAP kinase Fus3p. Both basal and stimulated phosphorylation occurred exclusively on serines. Mutagenesis of Clc1p was used to map major phosphorylation sites to serines 52 and 112, but conversion of all 14 serines in Clc1p to alanines [S(all)A] was necessary to eliminate phosphorylation. Cells expressing the S(all)A mutant Clc1p displayed no defects in Clc1p binding to clathrin heavy chain, clathrin trimer stability, sorting of a soluble vacuolar protein, or receptor-mediated endocytosis of mating pheromone. However, the trans-Golgi network membrane protein Kex2p was not optimally localized in mutant cells. Furthermore, pheromone treatment exacerbated the Kex2p localization defect and caused a corresponding defect in Kex2p-mediated maturation of the alpha-factor precursor. The results reveal a novel requirement for clathrin during the mating response and suggest that phosphorylation of the light chain subunit modulates the activity of clathrin at the trans-Golgi network.
Amino Acid Sequence↗