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W G Dunphy

Publications and source records attributed to W G Dunphy.

47 records · Page 3Linked to original sources
PubMed1986

Yeast and mammals utilize similar cytosolic components to drive protein transport through the Golgi complex.

Vesicular transport between successive compartments of the mammalian Golgi apparatus has recently been reconstituted in a cell-free system. In addition to ATP, transport requires both membrane-bound and cytosolic proteins. Here we report that the cytosol fraction from yeast will efficiently substitute for mammalian cytosol. Mammalian cytosol contains several distinct transport factors, which we have distinguished on the basis of gel filtration and ion-exchange chromatography. Yeast cytosol appears to contain the same collection of transport factors. Resolved cytosol factors from yeast and mammals complement each other in a synergistic manner. These findings suggest that the molecular mechanisms of intracellular protein transport have been conserved throughout evolution. Moreover, this hybrid cell-free system will enable the application of yeast genetics to the identification and isolation of cytosolic proteins that sustain intracellular protein transport.

Animals↗
PubMed1985

Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack.

Using monoclonal antibodies and electron microscopy, we have localized N-acetylglucosamine transferase I within the Golgi apparatus. This enzyme initiates the conversion of asparagine-linked oligosaccharides to the complex type. We have found that the enzyme is concentrated in the central (or medial) cisternae of the Golgi stack. Cisternae at the cis and trans ends of the Golgi complex appear to lack this protein. These experiments establish a function for the medial portion of the Golgi and imply that the Golgi is partitioned into at least three biochemically and morphologically distinct cisternal compartments.

Acetylglucosamine↗
PubMed1984

Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine.

Transport of the VSV-encoded glycoprotein (G protein) between successive compartments of the Golgi has been reconstituted in a cell-free system and is measured, in a rapid and sensitive new assay, by the coupled incorporation of 3H-N-acetylglucosamine (GlcNAc). This glycosylation occurs when G protein is transported during mixed incubations from the "donor" compartment in Golgi from VSV-infected CHO clone 15B cells (missing a key Golgi GlcNAc transferase) to the next, successive "acceptor" compartment (containing the GlcNAc transferase) in Golgi from wild-type CHO cells. Golgi fractions used in this assay have been extensively purified, and account for all of the donor and acceptor activity in the cells. Together with several other lines of evidence, this indicates that the cell-free system is highly specific, measuring only transport between sequential compartments in the Golgi stack. Transport in vitro is almost as efficient as in the cell, and requires ATP and the cytosol fraction in addition to protein components on the cytoplasmic surface of the Golgi membranes.

Acetylglucosamine↗
PubMed1983

Compartmentation of asparagine-linked oligosaccharide processing in the Golgi apparatus.

Golgi-associated processing of complex-type oligosaccharides linked to asparagine involves the sequential action of at least six enzymes. By equilibrium sucrose density gradient centrifugation of membranes from Chinese hamster ovary cells, we have partially resolved the set of four initial enzymes in the pathway (Mannosidase I, N-acetylglucosamine (GlcNAc) Transferase I, Mannosidase II, and GlcNAc Transferase II) from two later-acting activities (galactosyltransferase and sialyltransferase). In view of the recent demonstration that galactosyltransferase is restricted to the trans face of the Golgi complex in HeLa cells (Roth, J., and E.G. Berger, 1982, J. Cell Biol., 93:223-229), our results suggest that removal of mannose and attachment of peripheral N-acetylglucosamine may occur in some or all of the remaining cisternae on the cis side of the Golgi stack.

Animals↗
PubMed1982

Specific binding of phorbol ester tumor promoters to mouse tissues and cultured cells.

Phorbol esters bind to mouse tissues and chick embryo fibroblasts in a specific, saturable, and reversible fashion. The binding site, located in the membrane fraction, is heat and protease sensitive. Binding can be measured most readily with [3H]PDBu. Binding of [3H]PDBu is of high affinity and is inhibited competitively by nonradioactive phorbol esters; the dissociation constants of the phorbol esters correspond quantitatively to their respective biological and tumor-promoting activities. Of particular significance, highly inflammatory but weakly promoting or nonpromoting diterpene esters are much less potent than PMA. Binding of [3H]PMA has been measured directly. The results confirm that PMA and PDBu interact at the same major high-affinity binding site. [3H]PDBu binding is entropy driven. The equilibrium dissociation constant is independent of temperature, whereas the off-rate is highly temperature dependent. In vivo, specific binding activity increases during embryonic development. It also shows considerable variation among tissues. In the mouse, highest binding activity, 28 pmole/mg, is in the brain (skin, for example, binds 3.9 pmole/mg). Between regions of he brain, 10-fold differences in binding activity are found. The high level of phorbol ester binding in brain suggests that the phorbol ester receptor plays a functional rather than an exclusively information-transducing role in the cell. Growth of cells in the presence of PDBu causes marked down-modulation of phorbol ester receptors. In the GH4C1 rat pituitary cell line, receptor number is decreased by 80% in 24 hr. In membrane preparations, the phorbol ester binding affinity is calcium sensitive. It has been speculated that endogenous ligands interacting at the phorbol ester receptor may exist. The finding that the supernatant fraction from boiled or acidified brain inhibits [3H]PDBu binding is therefore exciting.

Animals↗
PubMed1981

Early and late functions associated with the Golgi apparatus reside in distinct compartments.

Enzymes that catalyze the two successive stages of Golgi-associated processing of asparagine-linked oligosaccharides distributed differently when membranes from Chinese hamster ovary cells were centrifuged in a sucrose density gradient. A mannosidase that removes only outer, alpha-1,2-linked mannose residues from the precursor oligosaccharides of the vesicular stomatitis viral G protein (to yield a "trimmed" oligosaccharide core) was separated from enzymes (galactosyl- and sialyltransferases) that act in the later, terminal stage of glycosylation. Freshly acylated G protein with newly trimmed oligosaccharides banded in the distribution of early-acting membranes, defined by the mannosidase, whereas G protein pulse-labeled with [3H]galactose distributed in the profile of the late-acting membranes. G protein present in the early-acting membranes in crude fractions could be terminally glycosylated by incubation with exogenous Golgi membranes in vitro; G protein lost its ability to be processed in vitro as it appeared to enter the late-acting membranes in vivo. These experiments reveal the existence of two distinct compartments through which intracellularly transported proteins such as G pass in sequence as Golgi-associated processes are carried out. It is likely that these compartments consist of cisternae on the cis and trans sides of the Golgi stack.

Animals↗
PubMed1981

Kinetics and subcellular localization of specific [3H]phorbol 12, 13-dibutyrate binding by mouse brain.

The specific binding of [3H]phorbol 12,13-dibutyrate ([3H]-PDBU) to particulate preparations from mouse brain has been further characterized. Kinetic analysis, using a filtration assay to measure binding, yielded a second-order rate constant at 23 degrees of 3.75 X 10(7) M-1 min-1 and a first-order dissociation rate constant of 0.21 min-1. The Kd of 5.6 nM calculated from the kinetic data agreed well with the value determined previously in equilibrium binding studies. The Kd for [3H]PDBU binding varied only slightly with temperature. From its temperature dependence, [3H]PDBU binding appeared to be associated with a small increase in enthalpy (delta H degrees = +0.4 kcal/mol) and a large increase in entropy (delta S degrees = +38 e.u.). Such values are characteristic for hydrophobic interactions. The dissociation rate constant for binding, in contrast to the Kd, varied dramatically with temperature. The half-time for release ranged from 1.75 min at 30 degrees to 62 min at 4 degrees. The Kd for binding was Ca2+ sensitive; chelation of Ca2+ by ethyleneglycolbis(beta-aminoethyl ether)N,N'-tetraacetic acid increased the Kd 2.4-fold. Upon subcellular fractionation, the specific [3H]PDBU binding activity was exclusively particulate; no binding to cytosol was detectable. Binding clearly did not correlate with nuclear or mitochondrial markers. On the other hand, a broader distribution of binding activity was seen on sucrose density gradients than for either Na+-K+-adenosine triphosphatase activity or binding of quinuclidinyl benzilate (a muscarinic cholinergic antagonist). The localization of specific [3H]PDBU binding to the plasma membrane therefore remains uncertain.

Animals↗
PubMed1980

Phorbol related esters have similar structure-activity relations in Swiss 3T3 cells and chicken embryo fibroblasts.

We have examined the activity of nine phorbol-related diterpene esters for stimulation of 2-deoxyglucose uptake in the Swiss 3T3 mouse cell line. The half-maximally effective doses (ED50's) of these derivatives ranged over four orders of magnitude. The potencies of the seven phorbol esters studied correlated well with their previously determined potencies in vitro for stimulation of 2-deoxyglucose uptake in chicken embryo fibroblasts and with thier potencies in vivo in the mouse ear inflammation assay. Two esters of 12-deoxyphorbol which are highly inflammatory but weakly promoting showed the same low potency in 3T3 cells as in chick embryo fibroblasts. These results suggest that 3T3 cells and chick embryo fibroblasts have homologous targets for the phorbol related diterpene esters. All phorbol related diterpene esters were of comparable efficacy in the 3T3 system with the exception of 4-O-methylphorbol 12-myristate 13-acetate. The lower efficacy of 4-O-methylphorbol 12-myristate 13-acetate may help account for its inactivity as a tumor promoter.

Animals↗
PubMed1980

Characterization of specific binding of [3H]phorbol 12,13-dibutyrate and [3H]phorbol 12-myristate 13-acetate to mouse brain.

[20-3H]Phorbol 12,13-dibutyrate ([3H]PDBU) and [20-3H]-phorbol 12-myristate 13-acetate ([3H]PMA) bound specifically and with high affinity to one class of saturable binding sites in particulate preparations from mouse brain. The dissociation constants for binding of [3H]PDBU and [3H]PMA were 7 nM and 66 pm, respectively. At half-maximal specific binding, binding of [3H]PDBU was 96% specific and binding of [3H]PMA was 91% specific. At saturation, 28 pmol of [3H]PDBU were bound per mg protein. Nonradioactive phorbol 12-myristate 13-acetate blocked > 95% of the specific [3H]PDBU binding. Conversely, nonradioactive phorbol 12,13-dibutyrate blocked > 95% of the specific [3H]PMA binding. The binding sites in mouse brain for [3H]PDBU and [3H]PMA thus appear to be equivalent. In addition to phorbol 12-myritate 13-acetate, seven other biologically active phorbol-related diterpenes inhibited [3H]PDBU binding with potencies which correlated with their biological activities. In the case of phorbol 12,13-diacetate, inhibition of [3H]PDBU binding was shown to be competitive. Phorbol (20 microgram/ml) and 4 alpha-phorbol 12,13-didecanoate (20 microgram/ml), two compounds devoid of biological activity, had no effect on binding. Binding was sensitive to heat, papain, and phospholipase A2 but was insensitive to neuraminidase. Based on the high level of specific phorbol ester binding in brain, we postulate that the phorbol ester target plays a functional rather than information-transducing role in the cell.

Animals↗