Hrs-2 regulates receptor-mediated endocytosis via interactions with Eps15.

Hrs-2, via interactions with SNAP-25, plays a regulatory role on the exocytic machinery. We now show that Hrs-2 physically interacts with Eps15, a protein required for receptor-mediated endocytosis. The Hrs-2/Eps15 interaction is calcium dependent, inhibited by SNAP-25 and alpha-adaptin, and results in the inhibition of receptor-mediated endocytosis. Immunoelectron microscopy reveals Hrs-2 localization on the limiting membrane of multivesicular bodies, organelles in the endosomal pathway. These data show that Hrs-2 regulates endocytosis, delineate a biochemical pathway (Hrs-2-Eps15-AP2) in which Hrs-2 functions, and suggest that Hrs-2 acts to provide communication between endo- and exocytic processes.

Substrate inhibition and affinities of the glyoxysomal beta-oxidation of sunflower cotyledons

During the glyoxysomal beta-oxidation of long-chain acyl-CoAs, short-chain intermediates accumulate transiently (Kleiter and Gerhardt 1998, Planta 206: 125-130). The studies reported here address the underlying factors. The studies concentrated upon the aspects of (i) chain length specificity and (ii) metabolic regulation of the glyoxysomal beta-oxidation of sunflower (Helianthus annuus L.) cotyledons. (i) Concentration-rate curves of the beta-oxidation of acyl-CoAs of various chain lengths showed that the beta-oxidation activity towards long-chain acyl-CoAs was higher than that towards short-chain acyl-CoAs at substrate concentrations <20 &mgr;M. At substrate concentrations >20 &mgr;M, long-chain acyl-CoAs were beta-oxidized more slowly than short-chain acyl-CoAs because the beta-oxidation of long-chain acyl-CoAs is subject to substrate inhibition which had already started at 5-10 &mgr;M substrate concentration and results from an inhibition of the multifunctional protein (MFP) of the beta-oxidation reaction sequence. However, low concentrations of free long-chain acyl-CoAs are rather likely to exist within the glyoxysomes due to the acyl-CoA-binding capacity of proteins. Consequently, the beta-oxidation rate towards a parent long-chain acyl-CoA will prevail over that towards the short-chain intermediates. (ii) Low concentrations (

A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.

We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY). Yeast spheroplasts are lysed by extrusion through polycarbonate filters. After differential centrifugation, a 125,000-g pellet is enriched for radiolabeled proCPY and is used as "donor" membranes. A 15,000-g pellet, harvested from nonradiolabeled cells and enriched for vacuoles, is used as "acceptor" membranes. When these membranes are incubated together with ATP and cytosolic extracts, approximately 50% of the radiolabeled proCPY is processed to mature CPY. Maturation was inhibited by dilution of donor and acceptor membranes during incubation, showed a 15-min lag period, and was temperature sensitive. Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from a PEP4 yeast strain, indicating intercompartmental transfer. Cytosol made from a yeast strain deleted for the VPS33 gene was less efficient at driving transport. Moreover, antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays. This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of a VPS gene product.

The vesicle transport protein Vps33p is an ATP-binding protein that localizes to the cytosol in an energy-dependent manner.

Molecular mechanisms of vesicle transport between the prevacuolar compartment and the vacuole in yeast or the lysosome in mammalian cells are poorly understood. To learn more about the specificity of this intercompartmental step, we have examined the subcellular localization of a SEC1 homologue, Vps33p, a protein implicated to function in transport between the prevacuolar compartment and the vacuole. Following short pulses, 80-90% of newly synthesized Vps33p cofractionated with a cytosolic enzyme marker after making permeabilized yeast cells. However, during a chase, 20-40% of Vps33p fractionated with permeabilized cell membranes in a time-dependent fashion with a half-time of approximately 40 min. Depletion of cellular ATP increased the association rate to a half-time of approximately 4 min and caused 80-90% of newly synthesized Vps33p to be associated with permeabilized cell membranes. The association of Vps33p with permeabilized cell membranes was reversible after restoring cells with glucose before permeabilization. The N-ethylmaleimide-sensitive fusion protein homologue, Sec18p, a protein with known ATP binding and hydrolysis activity, displayed the same reversible energy-dependent sedimentation characteristics as Vps33p. We determined that the photosensitive analog, 8-azido-[alpha-32P]ATP, could bind directly to Vps33p with low affinity. Interestingly, excess unlabeled ATP could enhance photoaffinity labeling of 8-azido-[alpha-32P]ATP to Vps33p, suggesting cooperative binding, which was not observed with excess GTP. Importantly, we did not detect significant photolabeling after deleting amino acid regions in Vps33p that show similarity to ATP interaction motifs. We visualized these events in living yeast cells after fusing the jellyfish green fluorescent protein (GFP) to the C terminus of full-length Vps33p. In metabolically active cells, the fully functional Vps33p-GFP fusion protein appeared to stain throughout the cytoplasm with one or two very bright fluorescent spots near the vacuole. After depleting cellular ATP, Vps33p-GFP appeared to localize with a punctate morphology, which was also reversible upon restoring cells with glucose. Overall, these data support a model where Vps33p cycles between soluble and particulate forms in an ATP-dependent manner, which may facilitate the specificity of transport vesicle docking or targeting to the yeast lysosome/vacuole.

Kinetic, binding, and NMR studies of perdeuterated yeast phosphoglycerate kinase and its interactions with substrates.

Perdeuterated yeast phosphoglycerate kinase (2HPGK) was prepared from yeast cells grown in 99.9% 2H2O and an acid hydrolysate from deuterated algal cells. Kinetic and binding studies suggested that perdeuterated enzyme was similar to the isotopically normal PGK. The use of 2HPGK not only eliminated the spectral overlap between the enzyme and substrate nuclear Overhauser effect (NOE) cross-peaks, but also permitted observation of weak transfer NOE cross-peaks between the substrate protons that are greater than 4 A apart. Intensity of NOE cross-peaks was used to determine the interproton distances of enzyme-bound Mg(II)dATP. These distances were then used in model building studies to determine the conformation of Mg(II)dATP. The average conformation of enzyme-bound dATP is anti with O4'/C2' endo ribose pucker and trans, gauche about the C4'-C5' bond. Although many spin diffusion pathways were eliminated by protein deuteration, spin diffusion was still observed between the protons of the substrate at mixing times longer than 25 ms.

Conformational changes in yeast phosphoglycerate kinase upon substrate binding.

Small-angle neutron scattering (SANS) was used to measure the radius of gyration (Rg) of solutions of phosphoglycerate kinase (PGK) in a variety of substrate environments in D2O. The Rg of 24.0 A was measured for native PGK. A decrease in Rg was observed for the following: 23.7 A for PGK+sulphate; 23.5 A for PGK+ beta, gamma-bidentate Cr(H2O)4ATP (CrATP); 23.3 A for PGK + 3-phospho-D-glycerate (PGA)+CrATP; 22.9 A for PGK+CrATP+sulphate; 22.6 A for PGK+PGA+CrATP+sulphate. The statistical error was about +/- 0.3 A, which is less than systematic effects in this system. These results are consistent with catalysis by a hinge-bending motion of the enzyme. Since CrATP is not hydrolyzed, these results represent the conformational states of the bound substrates in the catalytically relevant ternary complex in the absence of product formation. The second virial coefficient is also measured for this system and this is consistent with that calculated from the protein volume only.

Peroxisomal degradation of branched-chain 2-oxo acids.

Branched-chain 2-oxo acids which are formed by transamination of leucine, isoleucine, and valine are metabolized by the peroxisomes from mung bean (Vigna radiata L.) hypocotyls. Acylcoenzyme A (CoA) thio ester intermediates of the pathways were separated by reversed-phase high performance liquid chromatography. Retention time and cochromatography of individual acyl-CoA reference standards were used for identification of the acyl-CoA esters separated from the assay mixtures. Based on the results of identification and those of kinetic experiments, pathways of the peroxisomal degradation of 2-oxoisocaproate, 2-oxoisovalerate, and 2-oxo-3-methylvalerate are suggested.

Catalase Synthesis and Turnover during Peroxisome Transition in the Cotyledons of Helianthus annuus L.

Based on measurements of total catalase hematin and the degradation constants of catalase hematin, zero order rate constants for the synthesis of catalase were determined during the development of sunflower cotyledons (Helianthus annuus L.). Catalase synthesis reached a sharp maximum of about 400 picomoles hematin per day per cotyledon at day 1.5 during the elaboration of glyoxysomes in the dark. During the transition of glyoxysomes to leaf peroxisomes (greening cotyledons, day 2.5 to 5) catalase synthesis was constant at a level of about 30 to 40 picomoles hematin per day per cotyledon. In the cotyledons of seedlings kept in the dark (day 2.5 to 5) catalase synthesis did not exceed 10 picomoles hematin per day per cotyledon. During the peroxisome transition in the light, total catalase hematin was maintained at a high level, whereas total catalase activity rapidly decreased. In continuous darkness, total catalase hematin decreased considerably from a peak at day 2. The results show that both catalase synthesis and catalase degradation are regulated by light. The turnover characteristics of catalase are in accordance with the concept that glyoxysomes are transformed to leaf peroxisomes as described by the one population model and contradict the two population model and the enzyme synthesis changeover model which both postulate de novo formation of the leaf peroxisome population and degradation of the glyoxysome population.

Oxidative decarboxylation of branched-chain 2-oxo Fatty acids by higher plant peroxisomes.

Peroxisomes from mung bean (Vigna radiata L.) hypocotyls catalyze, in the presence of branched-chain 2-oxo fatty acid, CoASH and NAD, the release of CO(2), and the formation of NADH and acyl-CoA. The acyl-CoA contains one carbon atom less than the branched-chain 2-oxo fatty acid and serves as substrate for the peroxisomal acyl-CoA oxidase. CO(2) release, NADH and acyl-CoA formation occur in 1:1:1 stoichiometry. For the first time the data demonstrate directly the oxidative decarboxylation of branched-chain 2-oxo fatty acids in higher plants and a location of this activity in the peroxisomes.

Carnitine-acyltransferase activity of mitochondria from mung-bean hypocotyls.

Carnitine-acyltransferase activity assayed with acetyl-CoA, octanoyl-CoA, or palmitoyl-CoA is associated with the mitochondrial but not with the peroxisomes of mung-bean hypocotyls. Using mitochondria as an enzyme source, a half-maximal reaction rate is obtained with a palmitoyl-CoA concentration approximately twice that required with acetyl-CoA. In the presence of a saturating acetyl-CoA concentration the carnitine-acyltransferase activity is not enhanced by palmitoyl-CoA as additional substrate. However, palmitoylcarnitine is formed in addition to acetylcarnitine, and the formation of acetylcarnitine is competitively inhibited by palmitoyl-CoA. It is concluded that the mitochondria of mung-bean hypocotyls possess a carnitine acyltransferase of broad substrate specificity with respect to the chainlength of the acyl-CoA and that the demonstration of a carnitine-palmitoyltransferase activity in plant mitochondria does not indicate the presence of a specific carnitine long-chain acyltransferase.

Catalase Degradation in Sunflower Cotyledons during Peroxisome Transition from Glyoxysomal to Leaf Peroxisomal Function.

First order rate constants for the degradation (degradation constants) of catalase in the cotyledons of sunflower (Helianthus annuus L.) were determined by measuring the loss of catalase containing (14)C-labeled heme. During greening of the cotyledons, a period when peroxisomes change from glyoxysomal to leaf peroxisomal function, the degradation of glyoxysomal catalase is significantly (P = 0.05) slower than during all other stages of cotyledon development in light or darkness. The degradation constant during the transition stage of peroxisome function amounts to 0.205 day(-1) in contrast to the constants ranging from 0.304 day(-1) to 0.515 day(-1) during the other developmental stages. Density labeling experiments comprising labeling of catalase with (2)H(2)O and its isopycnic centrifugation on CsCl gradients demonstrated that the determinations of the degradation constants were not substantially affected by reutilization of (14)C-labeled compounds for catalase synthesis. The degradation constants for both glyoxysomal catalase and catalase synthesized during the transition of peroxisome function do not differ. This was shown by labeling the catalases with different isotopes and measuring the isotope ratio during the development of the cotyledons. The results are inconsistent with the concept that an accelerated and selective degradation of glyoxysomes underlies the change in peroxisome function. The data suggest that catalase degradation is at least partially due to an individual turnover of catalase and does not only result from a turnover of the whole peroxisomes.

Activation of fatty acids by non-glyoxysomal peroxisomes.

Peroxisomes from mung-bean hypocotyls catalyze, in the presence of fatty acids, CoASH, ATP, and MgCl2, the formation of acyl-CoA, AMP, and pyrophosphate in a 1:1:1 stoichiometry. This observation demonstrates that the peroxisomes of mung-bean hypocotyls possess an acyl-CoA synthetase (EC 6.2.1.3) for fatty-acid activation. Acyl-CoA synthetase activity is associated with the non-glyoxysomal peroxisomes from various tissues. The acyl-CoA synthetase of the peroxisomes of the mung-bean hypocotyl utilizes oleic, linoleic, and linolenic acid most effectively (3 nkat·mg(-1) peroxisomal protein). In contrast to the ß-oxidation enzymes of the peroxisomes whith are largely solubilized in the presence of 0.2 mol·l(-1) KCl, the acyl-CoA synthetase remains associated with the membrane fraction of peroxisomes. On the basis of the latency of the enzyme and its resistance to protease treatment of the peroxisomes, it is concluded that the enzyme is located at the matrix face of the peroxisome membrane.

Localization of ß-oxidation enzymes in peroxisomes isolated from nonfatty plant tissues.

Peroxisomes from spinach leaves, mungbean hypocotyls, and potato tubers catalyze a palmitoyl-CoA-dependent, KCN-insensitive O2 uptake. In the course of this reaction O2 is reduced to H2O2 in a 1:1 stoichiometry and palmitoyl-CoA oxidized, in a 1:1 stoichiometry, to a product serving as substrate for enoyl-CoA hydratase. These findings demonstrate the existence of a peroxisomal acyl-CoA oxidase in these tissues. Enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), and thiolase (EC 2.3.1.9) are also associated with the peroxisomes from mung-bean hypocotyls and potato tubers (as well as with spinach leaf peroxisomes as recently reported; Gerhardt 1981, FEBS Lett. 126, 71). The low activities of these enzymes in mitochondrial fractions seem to be due to contaminating peroxisomes since the ratio of ß-oxidation enzyme activities to catalase activity did not significantly differ between peroxisomal and mitochondrial fractions isolated on sucrose density gradients. The proof of localization of ß-oxidation enzymes in peroxisomes without glyoxysomal function leads to the concept that fatty-acid oxidation is a consistent basic function of the peroxisome in cells of higher plants.

Synthesis of Isocitrate Lyase in Sunflower Cotyledons during the Transition in Cotyledonary Microbody Function.

Density-labeling with 10 millimolar K(15)NO(3)/70% (2)H(2)O has been used to investigate isocitrate lyase synthesis during greening of sunflower (Helianthus annuus L.) cotyledons when the glyoxysomal enzyme activities sharply decline and the transition in cotyledonary microbody function occurs. A density shift of 0.0054 (kilograms per liter) was obtained for the profile of isocitrate lyase activity in the CsCl gradient with respect to the (1)H(2)O control. Quantitative evaluation of the density-labeling data indicates that about 50% of the isocitrate lyase activity present towards the end of the transition stage in microbody function is due to enzyme molecules newly synthesized during this stage.

L-lactate dehydrogenase from leaves of Capsella bursa-pastoris (L.) Med. : I. Identification and partial characterization.

By ammonium sulfate fractionation and gel filtration an enzyme preparation which catalyzed NAD(+)-dependent L-lactate oxidation (10(-4) kat kg(-1) protein), as well as NADH-dependent pyruvate reduction (10(-3) kat kg(-1) protein), was obtained from leaves of Capsella bursa-pastoris. This lactate dehydrogenase activity was not due to an unspecific activity of either glycolate oxidase, glycolate dehydrogenase, hydroxypyruvate reductase, alcohol dehydrogenase, or a malate oxidizing enzyme. These enzymes could be separated from the protein displaying lactate dehydrogenase activity by gel filtration and electrophoresis and distinguished from it by their known properties. The enzyme under consideration does not oxidize D-lactate, and reduces pyruvate to L-lactate (the configuration of which was determined using highly specific animal L-lactate dehydrogenase). Based on these results the studied Capsella leaf enzyme is classified as L-lactate dehydrogenase (EC 1.1.1.27). It has a Km value of 0.25 mmol l(-1) (pH 7.0, 0.3 mmol l(-1) NADH) for pyruvate and of 13 mmol l(-1) (pH 7.8, 3 mmol l(-1) NAD(+)) for L-lactate. Lactate dehydrogenase activity was also detected in the leaves of several other plants.

Apparent Catalase Synthesis in Sunflower Cotyledons during the Change in Microbody Function: A Mathematical Approach for the Quantitative Evaluation of Density-labeling Data.

Density-labeling with 10 mm K(15)NO(3)/70% (2)H(2)O has been used to investigate catalase synthesis in different developmental stages of sunflower (Helianthus annuus L.) cotyledons. A mathematical approach is introduced for the quantitative evaluation of the density-labeling data. The method allows, in the presence of preexisting enzyme activity, calculation of this synthesized activity (apparent enzyme synthesis) which results from the balance between actual enzyme synthesis and the degradation of newly synthesized enzyme at a given time. During greening of the cotyledons, when the catalase activity declines and the population of leaf peroxisomes is formed, the apparent catalase synthesis is lower than, or at best equal to, that occurring during a developmental stage when the leaf peroxisome population is established and catalase synthesis and degradation of total catalase are in equilibrium. This result suggests a formation, in fatty cotyledons, of the leaf peroxisomes by transformation of the glyoxysomes rather than by de novo synthesis.

[Studies on the change of microbody function in cotyledons of Helianthus annuus L]. / Untersuchungen zur Funktionsänderung der Microbodies in den Keimblättern von Helianthus annuus L.

The enzyme patterns in sunflower cotyledons indicate that the glyoxysomal function of microbodies is replaced by the peroxisomal function of these organelles during the transition from fat degradation to photosynthesis. The separation of the microbody population into glyoxysomes and peroxisomes during this transition period is reported. The mean difference in density between the activity peaks of glyoxysomal and peroxisomal marker enzymes on a sucrose gradient was calculated to be 0.007±0.004 g/cm(3) and turned out to be significant (t=7.8>4.04=t 5;0.01). The activity peak of catalase coincides with that of isocitrate lyase in early stages of development, but shifts to the activity peak of peroxisomal marker enzymes during the transition period. No isozymes of the catalase could be detected by gel electrophoresis in the microbodies with the two different functions.During the rise of the peroxisomal marker enzymes no synthesis of the common microbody marker, catalase, could be demonstrated using the inhibitor allylisopropylacetamide. Using D2) for density labeling of newly-formed catalase, no difference is observed between the density of catalase from cotyledons grown on 99.8% D2O during the transition period and the density of enzyme from cotyledons grown on H2O. The activity of particulate glycolate oxidase is reduced 30-50% by allylisopropylacetamide, but is not affected by D2O. The chlorophyll formation in the cotyledons is strongly inhibited by both substances.