Regulation of intracellular membrane transport.

A number of proteins that are necessary for membrane transport have been identified using cell-free assays and yeast genetics. Although our knowledge of transport mechanisms remains limited, common themes are clearly emerging. In particular, specific GTP-binding proteins appear to be involved, not only at all steps of membrane traffic but also at more than one check-point within each step. The ordered sequence of events occurring during vesicle formation, targeting and fusion may be regulated in a stepwise manner by specific GTP-dependent switches, which act as modular elements of the transport mechanism.

Membrane transport: Take your fusion partners.

Recent studies of how vesicles are targeted to fuse with specific membranes inside cells highlight a role for extended coiled-coil proteins in tethering partner membranes prior to formation of the 'SNARE complex' that mediates the fusion reaction. The tethering protein is recruited to membranes by a Rab family GTPase

Membrane transport: deciphering fusion.

Membrane fusion events that occur in yeast have been reconstituted with a minimal set of SNARE protein components. This system has been exploited to establish the syntax underlying specificity of intracellular fusion events from yeast to mammals.

Glutamate uptake occurs at an early stage of synaptic vesicle recycling.

Rapid membrane recycling in nerve terminals is required to maintain rapid synaptic transmission. Following the fusion of synaptic vesicles with synaptic plasma membranes, recycling can occur via clathrin-coated vesicles (CCVs) [1-3]. The fate of these vesicles is uncertain: they could simply uncoat and acquire other proteins from the cytosol to regenerate synaptic vesicles or they may fuse with endosomal structures from which synaptic vesicles could then bud. We have purified both CCVs and synaptic vesicles from rat brain, and measured the ability of these vesicle fractions to take up the excitatory neurotransmitter glutamic acid. We found that the normalized levels of glutamate uptake by the two types of vesicle were very similar. For each vesicle fraction, uptake required ATP and Cl- and could be fully inhibited by the specific vacuolar proton pump (v-ATPase) inhibitor concanamycin. We suggest that this ability to refill vesicles with neurotransmitter at the earliest intermediate on the recycling pathway - the CCV - may allow uncoated vesicles to immediately enter the releasable pool without sacrificing the quantal nature of neurotransmitter release.

Involvement of the endosomal autoantigen EEA1 in homotypic fusion of early endosomes.

In mammalian cells, fusion between early endocytic vesicles has been shown to require the ubiquitous intracellular fusion factors N-ethylmaleimide-sensitive factor (NSF) and alpha-SNAP, as well as a factor specific for early endosomes, the small GTPase Rab5 [1-3]. We have previously demonstrated an additional requirement for phosphatidylinositol 3-kinase (PI 3-kinase) activity [4]. The membrane association of early endosomal antigen 1 (EEA1), a specific marker of early endosomes [5,6], has recently been shown to be similarly dependent on PI 3-kinase activity [7], and we therefore postulated that it might be involved in endosome fusion. Here, we present evidence that EEA1 has an important role in determining the efficiency of endosome fusion in vitro. Both the carboxy-terminal domain of EEA1 (residues 1098-1411) and specific antibodies against EEA1 inhibited endosome fusion when included in an in vitro assay. Furthermore, depletion of EEA1, both from the membrane fraction used in the assay by washing with salt and from the cytosol using an EEA1-specific antibody, resulted in inhibition of endosome fusion. The involvement of EEA1 in endosome fusion accounts for the sensitivity of the endosome fusion assay to inhibitors of PI 3-kinase.

Characterization of MTMR3. an inositol lipid 3-phosphatase with novel substrate specificity.

Inositol lipids play key roles in many fundamental cellular processes that include growth, cell survival, motility, and membrane trafficking. Recent studies on the PTEN and Myotubularin proteins have underscored the importance of inositol lipid 3-phosphatases in cell function. Inactivating mutations in the genes encoding PTEN and Myotubularin are key steps in the progression of some cancers and in the onset of X-linked myotubular myopathy, respectively. Myotubularin-related protein 3 (MTMR3) shows extensive homology to Myotubularin, including the catalytic domain, but additionally possesses a C-terminal extension that includes a FYVE domain. We show that MTMR3 is an inositol lipid 3-phosphatase, with a so-far-unique substrate specificity. It is able to hydrolyze PtdIns3P and PtdIns3,5P2, both in vitro and when heterologously expressed in S. cerevisiae, and to thereby provide the first clearly defined route for the cellular production of PtdIns5P. Overexpression of a catalytically dead MTMR3 (C413S) in mammalian cells induces a striking formation of vacuolar compartments that enclose membranous structures that are highly concentrated in mutant proteins.

Endosomal localization and receptor dynamics determine tyrosine phosphorylation of hepatocyte growth factor-regulated tyrosine kinase substrate.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.

Inhibition of endosome fusion by wortmannin persists in the presence of activated Rab5.

Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5(Q79L) or of a xanthosine triphosphate-binding mutant, rab5(D136N), in the presence of the nonhydrolysable analogue XTPgammaS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPgammaS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5(Q79L), beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPgammaS.

Down-regulation of MET, the receptor for hepatocyte growth factor.

The ligand-dependent degradation of activated tyrosine kinase receptors provides a means by which mitogenic signalling can be attenuated. In many cell types the ligand-dependent degradation of the tyrosine kinase receptor Met is completely dependent on the activity of the 26S proteasome (Jeffers et al., 1997b). We now show that degradation also requires trafficking to late endosomal compartments and the activity of acid dependent proteases as determined by the effects of a dominant negative form of dynamin (K44A) and a vacuolar-ATPase inhibitor, concanamycin. We show that in the presence of the proteasome inhibitor lactacystin, Met fails to redistribute from the plasma membrane to intracellular compartments. This observation is most consistent with the interpretation that proteasome activity is required for Met internalization and only indirectly for its degradation.

Detection of thiol modification following generation of reactive nitrogen species: analysis of synaptic vesicle proteins.

S-nitrosylation is an important means of regulating the activity of proteins. We have developed a method which allows unbiased identification of thiol modified proteins within a complex mixture following NO generation, by taking advantage of the fact that prior nitrosylation will block subsequent modification of cysteine residues with 1-biotinamido-4-[4'-(maleimidomethyl)-cyclohexane-carboxamid o] butane (biotin-BMCC). Thiol modified proteins are reduced in intensity when revealed by blotting and overlay with avidin-horseradish peroxidase. In the case of a purified synaptic vesicle fraction we observe a high degree of enrichment of specific biotinylated proteins relative to homogenate. We find that thiol modification of proteins in the presence of NO donors is widespread, occurring in the majority of proteins that will react with biotin-BMCC. In a further development of this technique we have depleted the biotinylated proteins from solubilised synaptic vesicles using avidin-agarose and analysed the supernatants with a panel of antibodies. This has allowed us to identify SNARE proteins (soluble NSF attachment protein receptors) as potential targets for S-nitrosylation.

A comparative study of band 3 aggregation in erythrocyte membranes by melittin and other cationic agents.

The technique of laser flash-induced transient dichroism has been used to measure the rotational diffusion of eosin-labelled band 3 proteins in erythrocyte ghosts. A retardation in the mobility of band 3, measured subsequent to the addition of a variety of polyvalent cationic species, has been interpreted to reflect aggregation or 'clustering' of the protein in the plane of the membrane. A comparative study is reported between three such aggregators: melittin, polylysine and Zn2+, wherein their respective abilities to induce aggregation have been measured under varying conditions. Unlike that for melittin, band 3 aggregation by polylysine and Zn2+ is shown to be sensitive to proteolytic degradation of the membrane and to the ionic strength of the surrounding medium. Studies with fragments of melittin derived from its chymotryptic cleavage show the hydrophilic C-terminal 20-26 section to possess independent aggregating ability, but also the requirement of the 1-19 hydrophobic section to be attached in order to prevent reversibility by high ionic strength buffers. Melittin is also shown to have a unique ability to aggregate bacteriorhodopsin reconstituted into DMPC vesicles, which is partially retained by its 1-19 but not by its 20-26 fragment.

Cytoskeletal restraints of band 3 rotational mobility in human erythrocyte membranes.

The interaction of band 3 with cytoskeletal proteins was investigated in erythrocyte membranes by measuring the rotational mobility of band 3 using the method of transient dichroism. It was found that selective proteolysis of ankyrin, a protein known to link band 3 to the spectrin-actin network, had no significant effect on band 3 rotation. Incubating ghosts to 70 degrees C, at which temperature ankyrin is expected to be denatured, also had no effect. It thus appears probable that linkage of band 3 to the cytoskeleton via ankyrin does not act as a restraint on band 3 rotational motion. It is suggested that this is a consequence of flexibility in the cytoskeletal structure. In further investigations of the effect of heat treatment, a large enhancement of band 3 rotational mobility was found to result from incubation of intact cells for 1 h at 50 degrees C. This effect was not observed if ghosts were subjected to the same treatment, nor did it occur if the incubation of cells was performed at 47 degrees C. These findings, in combination with previous studies of band 3 rotational mobility, indicate that the interactions which restrain band 3 are likely to be more complex than commonly envisaged.

Leakage of internal markers from erythrocytes and lipid vesicles induced by melittin, gramicidin S and alamethicin: a comparative study.

The membrane-disruptive capacities of melittin, derivatised melittins, alamethicin and gramicidin S have been compared for the human erythrocyte membrane and lipid vesicles of three different compositions (phosphatidylcholine, 85% phosphatidylcholine/15% phosphatidylserine, and a lipid analogue of the outer leaflet of the human erythrocyte membrane). The sensitivity to ionic strength, divalent metal ions and polylysine of release of fluorescent markers from liposomes and of haemoglobin from intact erythrocytes has been assayed. Acetyl melittin was found to he more effective than melittin in lysing phosphatidylcholine and phosphatidylcholine/phosphatidylserine vesicles, somewhat less effective in the lipid analogue and markedly less effective in lysing erythrocytes. Succinyl melittin was non-haemolytic, but was able to lyse lipid vesicles at a high concentration. Ca2+ inhibited melittin haemolysis at high ionic strength (150 mM NaCl), but produced a more complex response of stimulation followed by inhibition at low ionic strength. In lipid vesicles, Ca2+ either stimulated melittin lysis or was ineffective. Zn2+ exerted effects similar to Ca2+ with lipid vesicles at approx. 10-fold lower concentration except that a weak inhibition was observed for the erythrocyte membrane lipid analogue at high ionic strength. Polylysine strongly inhibited haemolysis by melittin at low ionic strength, but was ineffective or stimulatory in lipid vesicle lysis. High phosphate concentration also inhibited melittin haemolysis, but again no corresponding effect could he found in any of the lipid vesicle systems. These disparities between effects of melittin on erythrocytes and lipid vesicles support the proposal that melittin-protein interactions are of consequence to its haemolytic action. Similar experiments were performed with gramicidin S and alamethicin in order to compare their lytic properties with those of melittin. It was found that each lysin exhibited its own individual pattern of sensitivity to lipid composition, ionic strength and inhibition by cations. It thus appears likely that the detailed molecular interactions responsible for lysis are significantly different for each of these three agents.

Met receptor dynamics and signalling.

The receptor for hepatocyte growth factor (HGF), Met, controls a programme of invasive growth that combines proliferation with various moto- and morphogenetic processes. This process is important for development and organ regeneration, but dysregulation in transformed tissues can contribute to cancer progression and metastasis. Acute stimulation of tissue culture cells with HGF leads to Met downregulation via degradation through an endocytic mechanism that also requires proteasome activity. Perturbation of Met trafficking on the endocytic pathway, either at the level of the internalisation step or during sorting at the early endosome, leads to altered signalling outputs. Ubiquitination of Met through the E3-ligase Cbl is required for receptor downregulation, and a mutant receptor defective in Cbl binding is able to transform cells. We discuss the hypothesis that some naturally occurring Met mutants implicated in cancer may transform cells owing to defects in their trafficking along the endosomal degradation pathway.

Phosphorylation of GDI and membrane cycling of rab proteins.

Membrane transport is known to be regulated by protein phosphorylation and by small GTPases of the rab family. Using specific antibodies, we have identified a 55 kDa phosphorylated protein which co-immunoprecipitated with the cytosolic forms of rab5 and other rab proteins. We demonstrate, on the basis of its mobility in two-dimensional electrophoresis gels and its immunological properties, that this protein is rab GDI (p55/GDI). We also found that, a minor fraction of p55/GDI is membrane associated, but, whilst also complexed with rab proteins, it is not phosphorylated. On the basis of these data we suggest that the cycling of rab proteins between membranes and cytosol is regulated by phosphorylation of p55/GDI.

Phosphatidylinositol 3-kinase regulation of fluid phase endocytosis.

Endocytosis of the fluid phase marker, horse radish peroxidase, into baby hamster kidney cells is inhibited by treatment of cells with the fungal metabolite wortmannin. The IC50 of approximately 5 nM is consistent with the well-described action of wortmannin upon phosphatidylinositol (PI) 3-kinase. Analysis of the kinetics of uptake indicates a > 50% decrease in the initial rate of marker internalisation, a concomitant decrease in the volume of the early endosome and an increased efficiency of recycling of that marker which is internalised. As PI 3-kinase binds to activated growth factor receptors our data suggest that receptor activation can be coupled to receptor internalisation (down regulation) by localising PI 3-kinase stimulation of endocytosis.

A long-lived state for influenza virus-erythrocyte complexes committed to fusion at neutral pH.

The low pH-induced fusion of influenza virus with intact erythrocyte plasma membranes is preceded by a delay time following pH reduction, that is itself pH- and temperature dependent. At 37 degrees C/pH 4.8, lipid mixing between virus and target membranes begins < 2 s after pH reduction, whereas at 4 degrees C/pH 4.8, fusion does not commence until > 10 min after pH reduction. We have found that within this time period at 4 degrees C, a population of virus acquires the capacity to subsequently undergo fusion with high efficiency at elevated temperatures and pH 7.4. Both the kinetics and the extent of this pH 7.4 fusion depend upon the time of pre-incubation at pH 4.8/4 degrees C. Incubation at pH 7.4/4 degrees C, following this pre-incubation does not result in fusion, but the capacity to fuse at pH 7.4/37 degrees C is retained for a time period exceeding 1 h. The longevity of this fusion committed state makes it amenable to biochemical and immunological analysis. We have shown that it is insensitive to dithiothreitol, neuraminidase and trypsin, but is incapacitated by thermolysin or protease K. We conclude that only the HA2 sub-unit of influenza haemagglutinin is a necessary protein component of later stages of the fusion pathway.

Electron transfer and conformation states in bovine cytochrome c oxidase.

The fluorophores 1,5-I-AEDANS and eosin maleimide bind to subunit III of bovine cytochrome c oxidase. Fluorescence lifetime measurements have been made of bound AEDANS under a number of conditions. It appears that the spatial relationship between this bound probe and metal centers is unaffected by the redox changes in the enzyme. Cyanide binding to CuA-modified cytochrome c oxidase during turnover suggests that reduction of cytochrome a leads to exposure of the cytochrome a3-CuB binuclear center to incoming ligands. These results are discussed in terms of a model describing the roles of cytochrome a and CuA in triggering the "closed" to "open" transition.

Nitrogen sparing and the catabolic hormones in patients nursed at an elevated ambient temperature following major surgery.

Fasted patients managed at an elevated ambient temperature following major surgery have reduced nitrogen excretion and body protein catabolism. To investigate the mechanism behind this three 24 h urine collections were made in 16 patients nursed for 48 h following aortobifemoral surgery on a Clinitron fluidized bed at 32 degrees C and analysed for total urinary nitrogen, cortisol and catecholamine excretion. Results were compared with a similar group of patients nursed throughout on a standard ITU bed at 22 degrees C. Patients managed at the elevated ambient temperature showed a significant reduction in the cumulative total urinary nitrogen (20.73 g +/- 6.42 v 28.95 g +/- 6.44; mean +/- S.D.; p less than 0.002) and cortisol excretion (1238 microg +/- 436 v 2197 microg +/- 844; mean +/- S.D.; p less than 0.001). Catecholamine excretion was also reduced but failed to achieve significance. There were significant correlations between cumulative total nitrogen excretion and both cortisol (r = +0.414; p = 0.02; n = 32) and noradrenaline (r = +0.369; p = 0.05; n = 32). These results confirm that the beneficial effect of an elevated ambient temperature on postoperative protein metabolism is brought about through a reduction in metabolic stress.

Post-operative fatigue - a real phenomenon attributable to the metabolic effects of surgery on body nutritional stores.

Ambulatory monitoring of activity was undertaken in 97 patients before and at intervals following surgery employing a lightweight recorder and sensors to monitor posture and movement over 24h periods. A subjective assessment of fatigue, anthropometric measurements and clinical details were noted prior to each recording. Results were assessed using multiple regression analysis. Few patients exhibited any subjective feeling of fatigue. However, objective assessment did show a reduction in activity, several of the changes correlating with factors related to surgical stress and post-operative nutritional depletion. Reduction in time standing was related to both weight change at 2 weeks (p < 0.005) and duration of surgery (p < 0.05). Increase in time spent lying correlated with muscle loss at 2 weeks (p < 0.005). Number of steps walked was only influenced by weight change at 2 weeks (p < 0.05). Reduction in post-operative mobility may be related to the metabolic consequences of the surgery and its effects on depletion of nutritional stores. Manipulation of the response and aggressive nutritional support might well reduce post-operative fatigue.