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Most of the mitochondrial proteome originates from nuclear genes and is transported into the mitochondria after synthesis in the cytosol. Complex machineries which maintain the specificity of protein import and [...]

Non-transferrin-bound iron, commonly found in the plasma of iron-overloaded individuals, permeates into cells via pathways independent of the transferrin receptor. This may lead to excessive cellular accumulation of labile iron followed by oxidative damage and eventually organ failure. Mitochondria are the principal destination of iron in cells and a primary site of prooxidant generation, yet their mode of acquisition of iron is poorly understood. Using fluorescent probes sensitive to iron or to reactive oxygen species, targeted to cytosol and/or to mitochondria, we traced the ingress of labile iron into these compartments by fluorescence microscopy and quantitative fluorimetry. We observed that 1) penetration of non-transferrin-bound iron into the cytosol and subsequently into mitochondria occurs with barely detectable delay and 2) loading of the cytosol with high-affinity iron-binding chelators does not abrogate iron uptake into mitochondria. Therefore, a fraction of non-transferrin-bound iron acquired by cells reaches the mitochondria in a nonlabile form. The physiological role of occluded iron transfer might be to confer cells with a 'safe and efficient cytosolic iron corridor' to mitochondria. However, such a mechanism might be deleterious in iron-overload conditions, because it could lead to surplus accumulation of iron in these critical organelles. transport; fluorescence; oxidative stress

SopE is a bacteriophage-encoded effector protein of Salmonella enterica serovar Typhimurium that is translocated into the cytosol of eukaryotic cells by a type III secretion system (TTSS) (W.-D. Hardt, H. Urlaub, and J. E. Galan, Proc. Natl. Acad. Sci. USA 95:2574-2579, 1998; M. W. Wood, R. Rosqvist, P. B. Mullan, M. H. Edwards, and E. E. Galyov, Mol. Microbiol. 22:327-338, 1996). In this study, we provide evidence that an unlinked gene carried within the Salmonella pathogenicity island 1 (SPI-1), invB (K. Eichelberg, C. Ginocchio, and J. E. Galan, J. Bacteriol. 176:4501-4510, 1994), is required for the secretion of SopE through the SPI-1 TTSS. Furthermore, far-Western blotting analysis shows that SopE directly interacts with InvB through a domain located at its amino terminus. We conclude that InvB is the TTSS-associated chaperone for SopE.

PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in Escherichia coli, the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and two pepN mutants, 9218 and DH50[alpha][DELTA]pepN. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in both pepN mutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in both pepN mutants. This decreased ability to cleave these substrates is pepN-specific as there is no reduction in the ability to hydrolyse exopeptidase substrates in E. coli mutants lacking other peptidases, pepA, pepB or pepE. PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in E. coli. Further in vitro studies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference of E. coli PepN to be Arg>Ala>Lys>Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress in E. coli, demonstrating a physiological role for this aminoendopeptidase under some stress conditions.

The circadian clock in mammals is located in the hypothalamic suprachiasmatic nucleus. At the core of the clock are molecular autofeedback loops associated with clock gene transcription. However, the mechanisms of circadian signal transduction are basically unknown. A recent report by Ikeda et al. provides new insights into the intracellular signaling pathways involved in conveying circadian clock information from the core loop to cellular functions. Cytosolic [Ca.sup.2+] is proposed to be a key substance linking the 'pendulum' to the 'hands' of the clock.

Hippocalcin is a neuronal calcium sensor protein that possesses a [Ca.sup.2+] /myristoyl switch allowing it to trans-locate to membranes. Translocation of hippocalcin in response to increased cytosolic [Ca.sup.2+] was examined in HeLa cells expressing hippocalcin--enhanced yellow fluorescent protein (EYFP) to determine the dynamics and [Ca.sup.2+] affinity of the [Ca.sup.2+] /myristoyl switch in living cells. [Ca.sup.2+]-free hippocalcin was freely diffusible, as shown by photobleaching and use of a photoactivable GFP construct. The translocation was dependent on binding of [Ca.sup.2+] by EF-hands 2 and 3. Using photolysis of NP-EGTA, the maximal kinetics of translocation was determined ([t.sub.1/2] = 0.9 s), and this was consistent with a diffusion driven process. Low intensity photolysis of NP-EGTA produced a slow [Ca.sup.2+] ramp and revealed that translocation of hippocalcin--EYFP initiated at around 180 nM and was half maximal at 290 nM. Histamine induced a reversible translocation of hippocalcin-EYFP. The data show that hippocalcin is a sensitive [Ca.sup.2+] sensor capable of responding to increases in intracellular [Ca.sup.2+] concentration over the narrow dynamic range of 200--800 nM free [Ca.sup.2+]

Ryanodine receptor (RyR) channels from mammalian cardiac and amphibian skeletal muscle were incorporated into planar lipid bilayers. Unitary [Ca.sup.2+] currents in the SR lumen-to-cytosol direction were recorded at 0 mV in the presence of caffeine (to minimize gating fluctuations). Currents measured with 20 mM lumenal [Ca.sup.2+] as exclusive charge carrier were 4.00 and 4.07 pA, respectively, and not significantly different. Currents recorded at 1-30 mM lumenal [Ca.sup.2+] concentrations were attenuated by physiological [[K.sup.+]] (150 mM) and [[Mg.sup.2+]] (1 mM), in the same proportion (~55%) in mammalian and amphibian channels. Two amplitudes, differing by ~35%, were found in amphibian channel studies, probably corresponding to [alpha] and [beta] RyR isoforms. In physiological [[Mg.sup.2+]], [[K.sup.+]], and lumenal [[Ca.sup.2+]] (1 mM), the [Ca.sup.2+] current was just less than 0.5 pA. Comparison of this value with the [Ca.sup.2+] flux underlying [Ca.sup.2+] sparks suggests that sparks in mammalian cardiac and amphibian skeletal muscles are generated by opening of multiple RyR channels. Further, symmetric high concentrations of [Mg.sup.2+] substantially reduced the current carried by 10 mM [Ca.sup.2+] (~40% at 10 mM [Mg.sup.2+]), suggesting that high [Mg.sup.2+] may make sparks smaller by both inhibiting RyR gating and reducing unitary current. KEY WORDS: ryanodine receptor * [Ca.sup.2+] release * sarcoplasmic reticulum

During retrovirus assembly, the polyprotein Gag directs protein multimerization, membrane binding, and RNA packaging. It is unknown whether assembly initiates through Gag-Gag interactions in the cytosol or at the plasma membrane. We used two fluorescence techniques--two-photon fluorescence resonance energy transfer and fluorescence correlation spectroscopy--to examine Rous sarcoma virus Gag-Gag and -membrane interactions in living cells. Both techniques provide strong evidence for interactions between Gag proteins in the cytoplasm. Fluorescence correlation spectroscopy measurements of mobility suggest that Gag is present in large cytosolic complexes, but these complexes are not entirely composed of Gag. Deletion of the nucleocapsid domain abolishes Gag interactions and membrane targeting. Deletion of the membrane-binding domain leads to enhanced cytosolic interactions. These results indicate that Gag-Gag interactions occur in the cytosol, are mediated by nucleocapsid domain, and are necessary for membrane targeting and budding. These methods also have general applicability to in vivo studies of protein-protein and -membrane interactions involved in the formation of complex macromolecular structures.

Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptidestimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction.

Adenosine exerts a spectrum of energy-preserving actions on the heart including negative chronotropic effects. The pathways leading to adenosine formation have remained controversial. In particular, although cytosolic 5'-nucleotidases can catalyze adenosine formation in cardiomyocytes, their contribution to the actions of adenosine has not been documented previously. We recently cloned two closely related AMP-preferring cytosolic 5'-nucleotidases (cN-IA and -IB); the A form predominates in the heart. In this study, we overexpressed pigeon cN-IA in neonatal rat cardiomyocytes using an adenovirus. cN-IA overexpression increased adenosine formation and release into the medium caused by simulated hypoxia and by isoproterenol in the absence and presence of inhibitors of adenosine metabolism. Adenosine release was not affected by an ecto-5'-nucleotidase inhibitor, [alpha],[beta]-methylene-ADP, but was affected by a nucleoside transporter, dipyridamole. The positive chronotropic effect of isoproterenol (130 [+ or -] 3 vs. 100 [+ or -] 4 beats/min) was inhibited (107 [+ or -] 3 vs. 94 [+ or -] 3 beats/min) in cells overexpressing cN-IA, and this was reversed by the addition of the adenosine receptor antagonist 8-(p-sulfophenyl)theophilline (120 [+ or -] 3 vs. 90 [+ or -] 4 beats/min). Our results demonstrate that overexpressed cN-IA can be sufficiently active in cardiomyocytes to generate physiologically effective concentrations of adenosine at its receptors. chronotropic effect; catecholamines; ATP metabolism

The intracellular transport of iron and its incorporation into organelles are poorly understood processes in eukaryotes and virtually unknown in parasitic protists. The transport of iron is of particular interest in trichomonads, which possess hydrogenosomes instead of mitochondria. The metabolic functions of hydrogenosomes, which contain a specific set of FeS proteins, entirely depend on iron acquisition. In this work the incorporation of iron into the cattle parasite Tritrichomonas foetus was monitored. Iron was efficiently taken up from [sup.59]Fe-nitrilotriacetic acid and accumulated in the cytosol (88.9%) and hydrogenosomes (4.7% of the total radioactivity). Using atomic absorption spectrophotometry, an unusually high steady-state iron concentration in hydrogenosomes was determined [54.4 [+ or -] 1.1 nmol Fe [(mg protein).sup.-1]. The concentration of iron in the cytosol was 13.4 [+ or -] 0.5 nmol Fe [(mg protein).sup.-1]. Qualitative analysis of incorporated iron was performed using native gradient PAGE. The majority of the 59Fe in the cytosol appeared as the labile-iron pool, which represents weakly bound iron associated with compounds of molecular mass ranging from 5000 to 30 000 Da. Ferritin was not observed in Tt. foetus, nor in two other anaerobic protists, Entamoeba histolytica and Giardia intestinalis. Analysis of Tt. foetus hydrogenosomes showed at least nine iron-binding compounds, which were absent in metronidazole-resistant mutants. The major iron-binding compound was identified as [2Fe-2S] ferredoxin of the adrenodoxin type.

Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of [Ca.sup.2+] from the ER, concomitant uptake of [Ca.sup.2+] into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial [Ca.sup.2+] signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates [Ca.sup.2+]-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650-800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its [beta]1 and [micro]1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its [beta]1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated [beta]1 compared with phosphorylated [micro]1. Once on the membrane, the [micro]1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of [micro]1 (and [micro]2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70-mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.

Synaptotagmin I (Syt I), a low-affinity [Ca.sup.2+]-binding protein, is thought to serve as the [Ca.sup.2+] sensor in the release of neurotransmitter. However, functional studies on the calyx of Held synapse revealed that the rapid release of neurotransmitter requires only approximately micromolar [[Ca.sup.2+]], suggesting that Syt I may play a more complex role in determining the high-affinity [Ca.sup.2+] dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinity [Ca.sup.2+]-dependent exocytic pathways and express Syt I. Using patch-clamp capacitance measurements to monitor secretion and the acute antisense deletion of Syt I from differentiated cells, we have shown that the rapid and the most [Ca.sup.2+]-sensitive pathway of exocytosis in rat melanotrophs requires Syt I. Furthermore, stimulation of the [Ca.sup.2+]-dependent exocytosis by cytosol dialysis with solutions containing [micro]M [[Ca.sup.2+]] was completely abolished in the absence of Syt I. Similar results were obtained by the preinjection of antibodies against the CAPS ([Ca.sup.2+]-dependent activator protein for secretion) protein. These results indicate that synaptotagmin I and CAPS proteins increase the probability of vesicle fusion at low cytosolic [[Ca.sup.2+]]. rat melanotrophs; exocytic module; membrane capacitance; calcium sensor

Indicator molecules for caspase-3 activation have been reported that use fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein (the donor) and enhanced yellow fluorescent protein (EYFP; the acceptor). Because EYFP is highly sensitive to proton ([H.sup.+]) and chloride ion ([Cl.sup.-]) levels, which can change during apoptosis, this indicator's ability to trace the precise dynamics of caspase activation is limited, especially in vivo. Here, we generated an [H.sup.+]- and [Cl.sup.-]--insensitive indicator for caspase activation, SCAT, in which EYFP was replaced with Venus, and monitored the spatio-temporal activation of caspases in living cells. Caspase-3 activation was initiated first in the cytosol and then in the nucleus, and rapidly reached maximum activation in 10 min or less. Furthermore, the nuclear activation of caspase-3 preceded the nuclear apoptotic morphological changes. In contrast, the completion of caspase-9 activation took much longer and its activation was attenuated in the nucleus. However, the time between the initiation of caspase-9 activation and the morphological changes was quite similar to that seen for caspase-3, indicating the activation of both caspases occurred essentially simultaneously during the initiation of apoptosis.

Research has been conducted on bovine retinal protein phosphatase 2A. The increased efficiency of phosducin dephosphorylation by this phosphatase in light and rhodopsin dephosphorylation by the phosphatase in darkness through the protein phosphatase 2A enzyme's translocation from the membrane fraction in darkness to the cytosolic fraction under intense light conditions has been investigated and the details are reported.

Divalent metal transporter 1 (DMT1) mediates apical iron uptake into duodenal enterocytes and also transfers iron from the endosome into the cytosol after cellular uptake via the transferrin receptor. Hence, mutations in DMT1 cause systemic iron deficiency and anemia. DMT1 mRNA levels are increased in the duodenum of iron-deficient animals. This regulation has been observed for DMT1 mRNA harboring an iron-responsive element (IRE) in its 3' UTR, but not for a processing variant lacking a 3'UTR IRE, suggesting that the IRE regulates the expression of DMT1 mRNA in response to iron levels. Here, we show that iron regulation of DMT1 involves the expression of a previously unrecognized upstream 5' exon (exon 1A) of the human and murine DMT1 gene. The expression of this previously uncharacterized 5' exon is tissue-specific and particularly prevalent in the duodenum and kidney. It adds an in-frame AUG translation initiation codon extending the DMT1 ORF by a conserved sequence of 29-31 amino acids. In combination with the IRE- and non-IRE variants in the 3'UTR, our results reveal the existence of four DMT1 mRNA isoforms predicting the synthesis of four different DMT1 proteins. We show that two regulatory regions, the 5' promoter/exon 1A region and the IRE-containing terminal exon participate in iron regulation of DMT1 expression, which operate in a tissue-specific way. These results uncover an unexpected complexity of DMT1 expression and regulation, with implications for understanding the physiology, cell biology, and pathophysiology of mammalian iron metabolism.

Research has been conducted on the membrane-bound metalloprotease FtsH. The ability of this metalloprotease to recognize membrane-embedded substrates and to initiate proteolysis at a C-terminal cytosolic tail has been investigated and the details are reported.

The authors reviews the publications on heterotetrameric clathrin adaptors linking clathrin coat with cargo transmembrane proteins. The topics of interest include the atomic structure if the adaptor-protein 2 core which contacts with the cargo proteins' cytosolic tails and with the membrane.

Research has been conducted on the type II secreted protein YopQ in Yersinia enterocolitica. The production, cytosolic accumulation and secretion of this protein have been investigated in various Y. enterocolitica serotypes via immunoblotting and the details are presented.

Activation of the phagocyte NADPH oxidase complex requires assembly of the cytosolic factors [p47.sup.PHOX], [p67.sup.PHOX], [p40.sup.PHOX], and Rac with the membrane-bound cytochrome [b.sub.558]. We recently established a direct interaction between [p67.sup.PHOX] and cytochrome [b.sub.558]. In the present study, we show that removal of the C-terminal domain of [p67.sup.PHOX] increased its binding to cytochrome [b.sub.558]. Whereas phosphorylated [p40.sup.PHOX] alone did not bind to cytochrome [b.sub.558], phosphorylated [p47.sup.PHOX] did, and, moreover, it allowed the binding of [p40.sup.PHOX] to the cytochrome. Furthermore, both increased the binding of [p67.sup.PHOX] to the cytochrome. Phosphorylated [p47.sup.PHOX] thus appears to increase the binding of [p67.sup.PHOX] to cytochrome [b.sub.558] by serving as an adapter, bringing [p67.sup.PHOX] into proximity with cytochrome [b.sub.558], whereas phosphorylated [p40.sup.PHOX] may increase the binding by inducing a conformational change that allows [p67.sup.PHOX] to interact fully with cytochrome [b.sub.558].

Research has been conducted on the ricin A chain, protein toxins entering mammalian cells via endocytosis. The hypothesis that the lysine paucity reduces the chance of ubiquitination and ubiquitin-mediated proteasomal degradation has been investigated and the results are reported.

Research has been conducted on the anthrax toxin which consists of the projective antigen (PA), lethal factor and edema factor. Results indicate that PA (sub)63 is responsible for the toxin's catalytic fragment delivery to the target cell cytosol.

Research has been conducted on the small GTPase Rac 1. The theory suggesting that Rac 1 is a membrane-targeting molecule for p67 (super)phox has been investigated via recombinant chimeric proteins constructed to join functional domains of p67 (super)phox and Rac 1 and expressed in Escherichia coli.

When expressed in tobacco cells, the catalytic subunit of the dimeric ribosome inactivating protein, ricin, is first inserted into the endoplasmic reticulum (ER) and then degraded in a manner that can be partially inhibited by the proteasome inhibitor clastolactacystin [beta]-lactone. Consistent with the implication of cytosolic proteasomes, degradation of ricin A chain is brefeldin A-insensitive and the polypeptides that accumulate in the presence of the proteasome inhibitor are not processed in a vacuole-specific fashion. Rather, these stabilized polypeptides are in part deglycosylated by a peptide:N-glycanase-like activity. Taken together, these results indicate that ricin A chain, albeit a structurally native protein, can behave as a substrate for ER to cytosol export, deglycosylation in the cytosol, and proteasomal degradation. Furthermore, retrotranslocation of this protein is not tightly coupled to proteasomal activity. These data are consistent with the hypothesis that ricin A chain can exploit the ER-associated protein degradation pathway to reach the cytosol. Although well characterized in mammalian and yeast cells, the operation of a similar pathway to the cytosol of plant cells has not previously been demonstrated.

Research has been conducted on the cold shock proteins. The results of the immunofluorescence microscopy and fusion of the cold shock protein to the green fluorescent protein indicate that cold shock proteins localize to the cytosolic regions surrounding the nucleoid in the Bacillus subtilis growing cells.

Research has been conducted on the cytosolic tails of the adhesion receptor integrin (alpha)IIb(beta)3. The dynamic and structural properties of these tails have been studied in aqueous solution via nuclear magnetic resonance spectroscopy.

Protein disulfide isomerase (PDI) has been found to act as a redox-dependent chaperone in the unfolding of cholera toxin in the endoplasmic reticulum (ER) lumen. This happens after the A chain of the toxin has been cleaved. A role for PDI is suggested in retrograde protein transport into the cytosol, and it can function as a novel type of chaperone. The binding and release of substrates would then be regulated by a redox cycle, not an ATPase cycle.

Research has been conducted on the cytosolic protein synaphin/complexin binding to syntaxin within the syntaxin and synapto-some-associated protein receptor (SNARE) complex. Results indicate that the synaphin promotes SNAREs to form pre-complexes which oligomerize into higher order structures.

Oligomeric tubulin in large transporting complex has been found to be transported via kinesin in squid giant axons. The movement of fluorescence was monitored with confocal laser scanning microscopy and fluorescence correlation spectroscopy after injection of the axons with fluorescence-labeled tubulin

The translocation of the alpha, epsilon and iota isozymes of protein kinase C (PKC) were investigated in particulate fractions from the cytosol during brief intervals of global ischemia as well as reperfusion of ischemic rat myocardium. In particular, ischemia resulted in the phosphorylation of 26-, 20-, and 17-kDa particulate-associated proteins. Findings indicate that these translocation of PKC isozymes in the ischemic and reperfused ischemic rat heart leads to the phosphorylation of specific particulate proteins.

Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in [Ca.sup.2+]-dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC (two [Ca.sup.2+] bound state) in complex with the N-terminal fragment of TnI ([TnI.sub.1-47]). The structure was solved by the single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic R factor of 22.2% ([R.sub.free] = 32.6%). The central, connecting a-helix observed in the structure of uncomplexed TnC ([TnC.sub.free]) is unwound at the center (residues Ala-87, Lys-88, Gly-89, Lys-90, and Ser-91) and bent by 90 [degrees] . As a result, TnC in the complex has a compact globular shape with direct interactions between the N- and C-terminal lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long [TnI.sup.1-47] [Alpha]-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-end of the [TnI.sub.1-47] [Alpha]-helix is bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between [Ca.sup.2+] receptors integrated with the other proteins (TnC in Tn) and isolated in the cytosol (calmodulin). The TnC/[TnI.sub.1-47] structure implies a mechanism of how Tn regulates the muscle contraction and suggests a unique [Alpha]-helical regulatory TnI segment, which binds to the N-lobe of TnC in its [Ca.sup.2+] bound conformation.

The effects of metabolite S-methyl N,N-diethylthiocarbamate (MeDTC) sulfone and sulfoxide on the cytosolic and mitochondrial forms of recombinant hepatic aldehyde dehydrogenase (ALDH) in humans were investigated. Recombinant human cytosolic and mitochondrial aldehyde dehydrogenase was expressed in Escherichia coli and purified to homogeneity to examine the mechanism by which disulfiram inhibits human ALDH. Results showed that MeDTC sulfoxide and sulfone are strong inhibitors of human ALDH and have the potential as proximal inhibitors of ALDH after disulfiram administration.

The presence of a nonmuscle form of capZ, a heterodimeric actin capping protein that blocks both actin filament assembly and disassembly at the fast growing filament ends, in human erythrocytes was investigated. CapZ is thought to be found in the cytosol and is not linked to the short actin filaments in the erythrocyte membrane skeleton. EcapZ was purified from erythrocyte cytosol and its biochemical and functional properties were characterized. Evidence showed that the erythrocyte actin filaments may be capped by adducin, a membrane skeleton protein, instead of capZ.

Translocation of ricin A chain to the cytosol has been proposed to take place from the endoplasmic reticulum (ER), but attempts to visualize ricin in this organelle have failed. Here we modified ricin A chain to contain a tyrosine sulfation site alone or in combination with N-glycosylation sites. When reconstituted with ricin B chain and incubated with cells in the presence of [[Na.sub.2].sup.35]S[O.sub.4], the modified A chains were labeled. The labeling was prevented by brefeldin A and ilimaquinone, and it appears to take place in the Golgi apparatus. This method allows selective labeling of ricin molecules that have already been transported retrograde to this organelle. A chain containing C-terminal N-glycosylation sites became core glycosylated, indicating retrograde transport to the ER. In part of the toxin molecules, the A chain was released from the B chain and translocated to the cytosol. The finding that glycosylated A chain was present in the cytosol indicates that translocation takes place after transport of the toxin to the ER.

The action of ruthenium red, a membrane-impermeant ryanodine receptor (RyR) modulator, and adenosine 3',5'-cyclic diphosphate ribose (cADPr), a cell-impermeant RyR modulator, were investigated by monitoring calcium (Ca2+) concentration in osteoclasts isolated from the long bones of rats. An attenuated increase in cytosolic Ca2+ concentration was elicited in the presence of ruthenium red and cADPr. A similar result was observed even when the cell membrane was hyperpolarized with valinomycin, suggesting that the cell membrane is the locus of action of the two modulators.

The role of atrial cytosol in the regulation of muscarinic potassium channels was investigated by testing the effects of inhibits of kinases and phosphatases that are involved in channel activity. Results showed that the cytosol contains a protein with a size ranging from 95-130 kDa that may be involved in rapid desensitization of the inwardly rectifying potassium current via a GTP-binding protein.

The enzyme hexokinase in the cytosol has important functions in glucose metabolism. It is proposed that hexokinase isoform I binds to the mitochondrial outer membrane of different cell types. Fluorophore-labeling studies in living cells showed that hexokinase localizes in the mitochondria. Immunocytochemical techniques also showed that presence of 2-deoxyglucose weakens the binding of hexokinase to mitochondria.

An alpha-toxin permeabilized acini has been introduced as an alternative permeabilization agent to streptolysin O (SLO)-permeabilized pancreatic acini to study regulated exocytosis in the exocrine pancreas. The characteristics of the two agents have been compared by measuring Ca2+-dependent amylase secretion from both types of permeabilized acini. The results showed a rapid rundown in the responsiveness of the SLO-permeabilized cell due to the loss of cytosol.

A study was conducted to investigate the effect of Ag+ on calcium fluxes that are mediated by retinal rod Na+/Ca2+/K+ exchanger in bovine rod outer segments (ROS) by measuring the fluorescence intensity of cytosolic free Ca2+. The results revealed that AG+ accelerated the high-velocity efflux activity of Ca2+ from ROS, resulting to the lowering of free Ca2+ concentration on the cytosolic surface of the exchanger protein.

In vivo tubulation of golgi membrane depends upon the presence of ATP and cytosolic protein factor. The inhibition of the cytosol-dependent tubulation by moderate trypsinization of isolated golgi membranes indicates that the interaction between the tubulation factor and protein receptor occurs on the cytoplasmic region of the golgi complex. The native molecular weight of tubulation activity is nearly 125,000 to 140,000 as revealed by gel filtration chromatography. The activity is susceptible to heat, is nondialyzable and precipitates in ammonium sulfate.

Squid axons were studied to determine the role of cytosolic factors such as soluble motor proteins and accessory factors in the microtubule-directed transport of vesicular organelles. Previous studies have shown that addition of cytosol to in vitro cultures of squid axon induced an increase in the number of organelles moving on microtubules per unit time. An evaluation of this finding was made possible with the discovery that casein prevents adhesion of organelles to the coverglass. The results showed that the movement of organelles to the plus-end of microtubules did not depend on soluble motor proteins and accessory factors in the cytosol.

A study was done on the 23-kilodalton protein which is a substrate of protein kinase C. It was shown that p23 show large primary structure homologies with the human proteins MRP14 and MRP8 which are expressed in large amounts during chronic inflammation. It also cross-reacted with monoclonal antibodies specific for these human proteins. The association of p23 with p7 results to the formation of a heterodimeric complex which are tolerant of trypsin. These properties closely resemble those of proteins belonging to the S100 family.

In gram-negative bacteria, coexpression of single- and multicomponent efflux pumps may result in multiplicative enhancement of the level of resistance against cytoplasmically acting antibiotics. Here, a simple model is presented to show that this cooperative effect can be accounted for only if substrate capture by the multicomponent efflux transporter occurs in the periplasm but not the cytosol.

Combination of diverse approaches including enzymatic assays, site-directed mutagenesis, amino acid sequence alignment, and structure comparisons were employed to gain insight into the yeast cytosolic thioredoxin system, which is composed of NADPH, thioredoxin reductase (ScTrxR1), and thioredoxin 1 (ScTrx1) or thioredoxin 2 (ScTrx2). The structural comparisons and amino acid alignments proposed a new classification that includes a larger number of enzymes with thioredoxin reductase activity, neglected in the low/high molecular weight classification.

The effect of NO modification on the functionality of human mitochondrial and cytosolic branched-chain aminotransferases (hBCATm and hBCATc, respectively) is examined. The results have shown distinct functional/mechanistic responses to S-nitrosoglutathione (GSNO) modification between BCAT isoforms and have offered comparisons between the BCAT proteins and the respective cytosolic and mitochondrial hTrx and hGrx proteins.

Research has been conducted on SecY-SecE cytosolic and transmembrane regions. The authors have investigated the physical contact sites of these translocase sununits in their cytosolic domains via the site-specific cross-linking experiments identifying these regions.