Search

The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions (pH = 6.3), ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane-binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Meusser et al.)

Lactoferricin B (LfcinB) is a cationic antimicrobial peptide, and its capacity to damage the bacterial plasma membrane is suggested to be a main factor in LfcinB's antimicrobial activity. However, the specific processes and mechanisms in LfcinB-induced membrane damage are unclear. In this report, using confocal laser-scanning microscopy, we examined the interaction of LfcinB with single Escherichia coli cells and spheroplasts containing the water-soluble fluorescent probe calcein in the cytoplasm. LfcinB induced rapid calcein leakage from single E. coli cells and from single spheroplasts, indicating that LfcinB interacts directly with the plasma membrane and induces its rapid permeabilization. The proton ionophore carbonyl cyanide m -chlorophenylhydrazone suppressed this leakage. Next, we used the single giant unilamellar vesicle (GUV) method to examine LfcinB's interaction with GUVs comprising polar lipid extracts of E. coli containing a water-soluble fluorescent probe, Alexa Fluor 647 hydrazide (AF647). We observed that LfcinB stochastically induces local rupture in single GUVs, causing rapid AF647 leakage; however, higher LfcinB concentrations were required for AF647 leakage from GUVs than from E. coli cells and spheroplasts. To identify the reason for this difference, we examined the effect of membrane potential on LfcinB-induced pore formation, finding that the rate of LfcinB-induced local rupture in GUVs increases greatly with increasing negative membrane potential. These results indicate that membrane potential plays an important role in LfcinB-induced local rupture of lipid bilayers and rapid permeabilization of E. coli plasma membranes. On the basis of these results, we discuss the mode of action of LfcinB's antimicrobial activity., (© 2019 Hossain et al.)

Cyclic nucleotide-gated (CNG) channels produce the initial electrical signal in mammalian vision and olfaction. They open in response to direct binding of cyclic nucleotide (cAMP or cGMP) to a cytoplasmic region of the channel. However, the conformational rearrangements occurring upon binding to produce pore opening ( i.e. gating) are not well understood. SthK is a bacterial CNG channel that has the potential to serve as an ideal model for structure-function studies of gating but is currently limited by its toxicity, native cysteines, and low open probability ( P o ). Here, we expressed SthK in giant Escherichia coli spheroplasts and performed patch-clamp recordings to characterize SthK gating in a bacterial membrane. We demonstrated that the P o in cAMP is higher than has been previously published and that cGMP acts as a weak partial SthK agonist. Additionally, we determined that SthK expression is toxic to E. coli because of gating by cytoplasmic cAMP. We overcame this toxicity by developing an adenylate cyclase-knockout E. coli cell line. Finally, we generated a cysteine-free SthK construct and introduced mutations that further increase the P o in cAMP. We propose that this SthK model will help elucidate the gating mechanism of CNG channels., (© 2019 Morgan et al.)

The type VI secretion system (T6SS) with diversified functions is widely distributed in pathogenic Proteobacteria. The IcmF (intracellular multiplication protein F) family protein TssM is a conserved T6SS inner membrane protein. Despite the conservation of its Walker A nucleotide-binding motif, the NTPase activity of TssM and its role in T6SS remain obscure. In this study, we characterized TssM in the plant pathogen Agrobacterium tumefaciens and provided the first biochemical evidence for TssM exhibiting ATPase activity to power the secretion of the T6SS hallmark protein, hemolysin-coregulated protein (Hcp). Amino acid substitutions in the Walker A motif of TssM caused reduced ATP binding and hydrolysis activity. Importantly, we discovered the Walker B motif of TssM and demonstrated that it is critical for ATP hydrolysis activity. Protein-protein interaction studies and protease susceptibility assays indicated that TssM undergoes an ATP binding-induced conformational change and that subsequent ATP hydrolysis is crucial for recruiting Hcp to interact with the periplasmic domain of the TssM-interacting protein TssL (an IcmH/DotU family protein) into a ternary complex and mediating Hcp secretion. Our findings strongly argue that TssM functions as a T6SS energizer to recruit Hcp into the TssM-TssL inner membrane complex prior to Hcp secretion across the outer membrane.

The β(2) subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe(2)(III)-Tyr(•)) that is essential for nucleotide reduction. The β(2) subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β'). Although only β is capable of iron binding and Tyr(•) formation, cells lacking β' are either dead or exhibit extremely low Tyr(•) levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β' is required for iron loading and Tyr(•) formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr(•)-reducing agent hydroxyurea. Transient induction of β' in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr(•) levels in β. Tyr(•) can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β' and is inhibited by adding an iron chelator prior to, but not after, β' supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr(•) levels and ββ' activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr(•) cofactor in RNR.

The plasma membrane of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid and diphenylhexatriene. Diphenylhexatriene anisotropy is a good reporter of global membrane order. The fluorescence lifetimes of trans-parinaric acid are particularly sensitive to the presence and nature of ordered domains, but thus far they have not been measured in yeast cells. A long lifetime typical of the gel phase (>30 ns) was found in wild-type (WT) cells from two different genetic backgrounds, at 24 and 30 °C, providing the first direct evidence for the presence of gel domains in living cells. To understand their nature and location, the study of WT cells was extended to spheroplasts, the isolated plasma membrane, and liposomes from total lipid and plasma membrane lipid extracts (with or without ergosterol extraction by cyclodextrin). It is concluded that the plasma membrane is mostly constituted by ordered domains and that the gel domains found in living cells are predominantly at the plasma membrane and are formed by lipids. To understand their composition, strains with mutations in sphingolipid and ergosterol metabolism and in the glycosylphosphatidylinositol anchor remodeling pathway were also studied. The results strongly indicate that the gel domains are not ergosterol-enriched lipid rafts; they are mainly composed of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins, suggesting an important role in membrane traffic and signaling, and interactions with the cell wall. The abundance of the sphingolipid-enriched gel domains was inversely related to the cellular membrane system global order, suggesting their involvement in the regulation of membrane properties.

Cytochromes P450 (P450s) incur phosphorylation. Although the precise role of this post-translational modification is unclear, marking P450s for degradation is plausible. Indeed, we have found that after structural inactivation, CYP3A4, the major human liver P450, and its rat orthologs are phosphorylated during their ubiquitin-dependent proteasomal degradation. Peptide mapping coupled with mass spectrometric analyses of CYP3A4 phosphorylated in vitro by protein kinase C (PKC) previously identified two target sites, Thr(264) and Ser(420). We now document that liver cytosolic kinases additionally target Ser(478) as a major site. To determine whether such phosphorylation is relevant to in vivo CYP3A4 degradation, wild type and CYP3A4 with single, double, or triple Ala mutations of these residues were heterologously expressed in Saccharomyces cerevisiae pep4Delta strains. We found that relative to CYP3A4wt, its S478A mutant was significantly stabilized in these yeast, and this was greatly to markedly enhanced for its S478A/T264A, S478A/S420A, and S478A/T264A/S420A double and triple mutants. Similar relative S478A/T264A/S420A mutant stabilization was also observed in HEK293T cells. To determine whether phosphorylation enhances CYP3A4 degradation by enhancing its ubiquitination, CYP3A4 ubiquitination was examined in an in vitro UBC7/gp78-reconstituted system with and without cAMP-dependent protein kinase A and PKC, two liver cytosolic kinases involved in CYP3A4 phosphorylation. cAMP-dependent protein kinase A/PKC-mediated phosphorylation of CYP3A4wt but not its S478A/T264A/S420A mutant enhanced its ubiquitination in this system. Together, these findings indicate that phosphorylation of CYP3A4 Ser(478), Thr(264), and Ser(420) residues by cytosolic kinases is important both for its ubiquitination and proteasomal degradation and suggest a direct link between P450 phosphorylation, ubiquitination, and degradation.

In numerous cell types, tumoral cells, proliferating cells, bacteria, and yeast, respiration is inhibited when high concentrations of glucose are added to the culture medium. This phenomenon has been named the "Crabtree effect." We used yeast to investigate (i) the short term event(s) associated with the Crabtree effect and (ii) a putative role of hexose phosphates in the inhibition of respiration. Indeed, yeast divide into "Crabtree-positive," where the Crabtree effect occurs, and "Crabtree-negative," where it does not. In mitochondria isolated from these two categories of yeast, we found that low, physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulated the respiratory flux and that this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself was able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Using permeabilized spheroplasts from Crabtree-positive yeast, we have shown that the sole effect observed at physiological concentrations of hexose phosphates is an inhibition of oxidative phosphorylation by F16bP. This F16bP-mediated inhibition was also observed in isolated rat liver mitochondria, extending this process to mammalian cells. From these results and taking into account that F16bP is able to accumulate in the cell cytoplasm, we propose that F16bP regulates oxidative phosphorylation and thus participates in the establishment of the Crabtree effect.

The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.

The mechanism of lipopolysaccharide (LPS) transport in Gram-negative bacteria from the inner membrane to the outer membrane is largely unknown. Here, we investigated the possibility that LPS transport proceeds via a soluble intermediate associated with a periplasmic chaperone analogous to the Lol-dependent transport mechanism of lipoproteins. Whereas newly synthesized lipoproteins could be released from spheroplasts of Escherichia coli upon addition of a periplasmic extract containing LolA, de novo synthesized LPS was not released. We demonstrate that LPS synthesized de novo in spheroplasts co-fractionated with the outer membranes and that this co-fractionation was dependent on the presence in the spheroplasts of a functional MsbA protein, the protein responsible for the flip-flop of LPS across the inner membrane. The outer membrane localization of the LPS was confirmed by its modification by the outer membrane enzyme CrcA (PagP). We conclude that a substantial amount of LPS was translocated to the outer membrane in spheroplasts, suggesting that transport proceeds via contact sites between the two membranes. In contrast to LPS, de novo synthesized phospholipids were not transported to the outer membrane in spheroplasts. Apparently, LPS and phospholipids have different requirements for their transport to the outer membrane.

Contrary to what is widely believed, recent published results show that H2O2 does not freely diffuse across biomembranes. The fast removal of H2O2 by antioxidant enzymes is able to generate a gradient if H2O2 is produced in a different compartment from that containing the enzymes (Antunes, F., and Cadenas, E. (2000) FEBS Lett. 475, 121-126). In this work, we extended these studies and tested whether an active regulation of biomembranes permeability characteristics is part of the cell response to oxidative stress. Using Saccharomyces cerevisiae as a model, we showed that: (a) H2O2 gradients across the plasma membrane are formed upon exposure to external H2O2; (b) there is a correlation between the magnitude of the gradients and the resistance to H2O2; (c) there is not a correlation between the intracellular capacity to remove H2O2 and the resistance to H2O2; (d) the plasma membrane permeability to H2O2 decreases by a factor of two upon acquisition of resistance to this agent by pre-exposing cells either to nonlethal doses of H2O2 or to cycloheximide, an inhibitor of protein synthesis; and (e) erg3Delta and erg6Delta mutants, which have impaired ergosterol biosynthesis pathways, show higher plasma membrane permeability to H2O2 and are more sensitive to H2O2. Altogether, the regulation of the plasma membrane permeability to H2O2 emerged as a new mechanism by which cells respond and adapt to H2O2. The consequences of the results to cellular redox compartmentalization and to the origin and evolution of the eukaryotic cell are discussed.

DsbB is an Escherichia coli plasma membrane protein that reoxidizes the Cys30-Pro-His-Cys33 active site of DsbA, the primary dithiol oxidant in the periplasm. Here we describe a novel activity of DsbB to induce an electronic transition of the bound ubiquinone molecule. This transition was characterized by a striking emergence of an absorbance peak at 500 nm giving rise to a visible pink color. The ubiquinone red-shift was observed stably for the DsbA(C33S)-DsbB complex as well as transiently by stopped flow rapid scanning spectroscopy during the reaction between wild-type DsbA and DsbB. Mutation and reconstitution experiments established that the unpaired Cys at position 44 of DsbB is primarily responsible for the chromogenic transition of ubiquinone, and this property correlates with the functional arrangement of amino acid residues in the neighborhood of Cys44. We propose that the Cys44-induced anomaly in ubiquinone represents its activated state, which drives the DsbB-mediated electron transfer.

The major outer membrane lipoprotein (Lpp) of Escherichia coli requires LolA for its release from the cytoplasmic membrane, and LolB for its localization to the outer membrane. We examined the significance of the LolA-LolB system as to the outer membrane localization of other lipoproteins. All lipoproteins possessing an outer membrane-directed signal at the N-terminal second position were efficiently released from the inner membrane in the presence of LolA. Some lipoproteins were released in the absence of externally added LolA, albeit at a slower rate and to a lesser extent. This LolA-independent release was also strictly dependent on the outer membrane sorting signal. A lipoprotein-LolA complex was formed when the release took place in the presence of LolA, whereas lipoproteins released in the absence of LolA existed as heterogeneous complexes, suggesting that the release and the formation of a complex with LolA are distinct events. The release of LolB, an outer membrane lipoprotein functioning as the receptor for a lipoprotein-LolA complex, occurred with a trace amount of LolA, and therefore was extremely efficient. The LolA-dependent release of lipoproteins was found to be crucial for the specific incorporation of lipoproteins into the outer membrane, whereas lipoproteins released in the absence of LolA were nonspecifically and inefficiently incorporated into the membrane. The outer membrane incorporation of lipoproteins including LolB per se was dependent on LolB in the outer membrane. From these results, we conclude that lipoproteins in E. coli generally utilize the LolA-LolB system for efficient release from the inner membrane and specific localization to the outer membrane.

Candida albicans is the predominant species of yeast isolated from patients with oral candidiasis, which is frequently a symptom of human immunodeficiency virus infection and is a criterion for staging and progression of AIDS. Salivary histatins (Hsts) are potent in vitro antifungal agents and have great promise as therapeutic agents in humans with oral candidiasis. The molecular mechanisms by which Hsts kill yeast cells are not known. We report here, that unlike other antimicrobial proteins, Hsts do not display lytic activities to lipid membranes, measured by release and dequenching of the fluorescent dye calcein. Analysis of the magnitude and time course of Hst-induced calcein release from C. albicans cells further showed that loss of cell integrity was a secondary effect following cell death, rather than the result of primary disruption of the yeast cell membrane. 125I-Hst 5 binding studies indicated that C. albicans expressed a class of saturable binding sites (KD = 1 microM), numbering 8.6 x 10(5) sites/cell. Both Hst 3 and Hst 4 competed for these binding sites with similar affinities, which is consistent with the micromolar concentration of Hsts required for candidacidal activity. Specific 125I-Hst 5 binding was not detected to C. albicans spheroplasts, which were 14-fold less susceptible to Hst 5 killing, compared with intact cells in candidacidal assays. In overlay experiments, 125I-Hst 5 bound to a 67-kDa protein detected in C. albicans whole cell lysates and crude membrane fractions, but not in the yeast cell wall fraction. Consistent with the overlay data, cross-linking of 125I-Hst 5 to C. albicans resulted in the appearance of a specific 73-kDa 125I-Hst 5-containing complex that was not detected in the cell wall. 125I-Hst 5-binding protein of similar size was also observed in susceptible S. cerevisiae strain TI#20. This is the first description of Hst 5 binding sites on C. albicans which mediate cell killing and identification of a 67-kDa yeast Hst 5-binding protein. The binding characteristics of Hst 5 are in agreement with the observed potency of its biological effect and provide crucial information to the use of Hst 5 as a therapeutic agent. The presence of a specific C. albicans Hst 5-binding protein provides further insight into the potential mechanism of yeast killing and suggests a basis for differential activity between yeast killing and the nontoxic nature of Hsts to humans.

We have shown previously that the first transmembrane segment of leader peptidase can function to translocate the polar amino-terminal Pf3 domain across the membrane into the periplasm independently of the proton motive force (pmf) (Lee, J. I., Kuhn, A., and Dalbey, R. E. (1992) J. Biol. Chem. 267, 938-943). We now show that when the first transmembrane segment lacks a strong hydrophobic character, the pmf is required for translocation. In addition, we find that the amino-terminal acidic residue proximal to the transmembrane domain plays a critical role in pmf-dependent amino-terminal translocation. Moreover, the pmf is required to hold the amino-terminal domain in the periplasm to prevent it from slipping such that the amino terminus is no longer exposed to the periplasm. In all cases, translocation occurs under conditions in which the function of the Sec machinery is impaired. These studies show that the low hydrophobicity of the first apolar domain (the translocation signal) can be compensated for by a negative charge in the amino-terminal region, upon which the pmf acts.

The multidrug resistance-associated protein (MRP) is a member of the ATP binding cassette superfamily of transporters which includes the mammalian P-glycoproteins (P-gp) family. In order to facilitate the biochemical and genetic analyses of MRP, we have expressed human MRP in the yeast Saccharomyces cerevisiae and have compared its functional properties to those of the mouse Mdr3 P-gp isoform. Expression of both MRP and Mdr3 in the anthracycline hypersensitive mutant VASY2563 restored cellular resistance to Adriamycin in this mutant. MRP and Mdr3 expression produced pleiotropic effects on drug resistance in this mutant, as corresponding VASY2563 transformants also acquired resistance to the anti-fungal agent FK506 and to the K+/H+ ionophore valinomycin. The appearance of increased cellular resistance to the toxic effect of Adriamycin (ADM) in MRP and Mdr3 transformants was concomitant with a reduced intracellular accumulation of [14C]ADM in spheroplasts prepared from these cells. Moreover, MRP and Mdr3, but not control spheroplasts, could mediate a time-dependent reduction in the overall cell-associated [14C]ADM from preloaded cells, suggesting the presence of an active ADM transport mechanism in MRP and Mdr3 transformants. Finally, human MRP was found to complement the biological activity of the yeast peptide pheromone transporter Ste6 and partially restored mating in a sterile ste6 null mutant. These findings suggest that despite their relatively low level of structural homology, MRP and P-gp share similar functional aspects, since both proteins can mediate transport of chemotherapeutic drugs and the a mating peptide pheromone in yeast.

The sequence of biosynthetic steps from phosphatidylserine to phosphatidylethanolamine (via decarboxylation) and then phosphatidylcholine (via methylation) is linked to the intracellular transport of these aminoglycerophospholipids. Using a [3H]serine precursor and permeabilized yeast cells, it is possible to follow the synthesis of each of the aminoglycerophospholipids and examine the requirements for their interorganelle transport. This experimental approach reveals that in permeabilized cells newly synthesized phosphatidyl-serine is readily translocated to the locus of phosphatidylserine decarboxylase 1 in the mitochondria but not to the locus of phosphatidylserine decarboxylase 2 in the Golgi and vacuoles. Phosphatidylserine transport to the mitochondria is ATP independent and exhibits no requirements for cytosolic factors. The phosphatidylethanolamine formed in the mitochondria is exported to the locus of the methyltransferases (principally the endoplasmic reticulum) and converted to phosphatidylcholine. The export of phosphatidylethanolamine requires ATP but not any other cytosolic factors and is not obligately coupled to methyltransferase activity. The above described lipid transport reactions also occur in permeabilized cells that have been disrupted by homogenization, indicating that the processes are extremely efficient and may be dependent upon stable structural elements between organelles.

Vacuolar membrane vesicles were isolated from Candida albicans protoplasts, and marker enzyme assays were employed to identify the membranes as vacuolar in origin. The mechanisms of Ca2+ uptake and Ca2+ release at the vacuolar membrane were investigated. Ca2+ accumulation by vacuolar membrane vesicles can be generated via H+/Ca2+ antiport. The inside-acid pH is in turn generated by a vacuolar-type H(+)-ATPase, as demonstrated by the sensitivity of Ca2+ uptake to ionophores and the vacuolar H(+)-ATPase inhibitor bafilomycin A1. Vacuolar membrane vesicles exhibit two Ca2+ release pathways: one induced by inositol 1,4,5-trisphosphate (InsP3) and the other by inside-positive voltage. These two pathways are distinct with respect to the amount of Ca2+ released, the nature of response to successive stimuli, and their respective pharmacological profiles. The InsP3-gated pathway exhibits a K0.5 for InsP3 of 2.4 microM but is not activated by inositol 4,5-bisphosphate or inositol 1,3,4,5-tetrakisphosphate at concentrations up to 50 microM. Ca2+ release by InsP3 is blocked partially by low molecular weight heparin. Ca2+ released by the voltage-sensitive pathway occurs at membrane potentials estimated to be over a physiological range from 0 to 80 mV. The voltage-sensitive Ca2+ release pathway can be blocked by lanthanide ions and organic channel blockers such as ruthenium red and verapamil. Furthermore, the voltage-sensitive Ca2+ release pathway exhibits Ca(2+)-induced Ca2+ release. These findings are discussed in relation to the mechanism of Ca(2+)-mediated cellular signaling in C. albicans and other fungi.

To investigate the function of the membrane anchor region of a mammalian glycosyltransferase in yeast we constructed a fusion gene that encodes the 34 amino-terminal residues of rat liver beta-galactoside alpha-2,6-sialyl-transferase (EC 2.4.99.1) (ST) fused to the mature form of yeast invertase. Transformants of Saccharomyces cerevisiae expressing the fusion gene produced an intracellular heterogeneously N-glycosylated fusion protein of intermediate molecular weight between the core and fully extended N-glycosylated form of invertase, suggesting a post-endoplasmic reticulum (ER) localization. In two types of cell fractionation using sucrose density gradients the ST-invertase fusion protein cofractionated with Golgi marker proteins, whereas a minor fraction (about 30%) comigrated with a vacuolar marker; ST-invertase was not detected in other cell fractions including the ER and the plasma membrane. Consistent with Golgi localization, about 70% of the total amount of the ST-invertase fusion was immunoprecipitated with an antibody directed against alpha-1,6-mannose linkages. The results demonstrate that the membrane anchor region of a mammalian type II glycosyltransferase is able to target a protein to the secretory pathway and to a Golgi compartment of the yeast S. cerevisiae, indicating conservation of targeting mechanisms between higher and lower eukaryotes. Since typical yeast Golgi localization signals are missing in the ST-membrane anchor region the results also suggest that yeast as mammalian cells utilize diverse mechanisms to direct proteins to the Golgi.

The CorA Mg2+ transport systems of Salmonella typhimurium and Escherichia coli mediate both influx and efflux of Mg2+. The product of the CorA locus is sufficient for mediation of Mg2+ influx while product(s) of the unlinked CorBCD loci allow CorA to mediate efflux in addition to influx. The nucleotide sequences of the S. typhimurium and E. coli CorA loci have been determined. The locus in each species consists of a single gene expressing a protein with gel molecular masses of 42 kDa (S. typhimurium) and 39 kDa (E. coli). The predicted amino acid sequences of these proteins are each 316 amino acids in length, are 98% identical, and lack homology to any known protein. Although CorA is an integral membrane protein by biochemical criteria, its predicted amino acid sequence contains 28% charged amino acid. Membrane localization of CorA was shown to be dependent on the Sec pathway in E. coli. Hydropathy analysis predicts two C-terminal hydrophobic sequences of sufficient length to span the membrane bilayer. The membrane topology of CorA was determined by constructing deletion derivatives of CorA and genetically fusing them to BlaM or LacZ cassettes. The enzymatic activities of these hybrid proteins indicate that the N-terminal 235 amino acid residues of the CorA protein are located within the periplasmic space, comprising a single periplasmic domain. The C-terminal region of CorA is composed of three membrane-spanning segments rather than the two suggested by hydropathy plots, thus depositing the C terminus within the cytoplasm. This topology suggests that CorA functions as an oligomer since three membrane loops are most likely insufficient for any sort of membrane pore or channel. Its lack of homology to known proteins and its topology indicate that the CorA Mg2+ transporter represents a new class of membrane transport system.

Proton translocation in spheroplasts from Escherichia coli has been studied in two mutants, one of which expresses cytochrome o and the other cytochrome d as the terminal oxidase. Using the O2 pulse method, the H+/e- ratio of proton translocation associated with cytochrome o was confirmed to be near 2 at neutral pH, but was found to decrease considerably when the medium pH was raised above 8. At high pH there was an increase in H+/OH- permeability of the cell membrane, but this was not sufficient to explain the decline in proton ejection. The pH effect was confined to cytochrome o-linked activity. It was not present when cytochrome d generated the electrochemical proton gradient. This makes it improbable that the Na+/H+ antiporter is responsible. The most likely explanation for our finding is that there is a "slip" in the proton-pumping mechanism of cytochrome o at high pH.

The vacuole of the yeast Saccharomyces cerevisiae contains a proton-translocating ATPase that acidifies the vacuolar lumen and generates a pH gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of the genes encoding the A, B, or c subunit of the vacuolar ATPase are unable to acidify their vacuoles. These vat mutant strains accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. The kinetics of secretion suggests that missorting occurs in the Golgi complex or in post-Golgi vesicles. The presence of mature forms of the vacuolar proteins within the cell indicates that vat mutations do not cause defects in zymogen processing. Precursor forms of the membrane-associated vacuolar hydrolase alkaline phosphatase are also accumulated in vat mutant cells but to a lesser extent, suggesting that sorting of vacuolar membrane proteins is less sensitive to changes in the lumenal pH. A similar type of missorting defect can be induced in wild-type cells at pH 7.5. These results indicate that acidification of the vacuolar system is important for efficient sorting of proteins to the vacuole.

Postpolysomal extracts have been prepared from wild type haploid Saccharomyces cerevisiae cells (wt A364A) and from a temperature-sensitive mutant strain (ts 187, gene prt 1). The extracts, prepared via spheroplasts and depleted of endogenous mRNA with nuclease, translate exogenous natural mRNA and polyuridylic acid. The activity of wt A364A with respect to translation of yeast mRNA, poly(U)-dependent synthesis of polyphenylalanine which measures elongation components, reactions involved in the initiation of protein synthesis, and termination and release of polypeptides, is not significantly affected when spheroplasts are incubated at 39 degrees C for relatively short periods of time, prior to the preparation of the cell-free system. With extracts obtained from ts 187 cells, preincubation of spheroplasts at 39 degrees C prior to the preparations of the cell-free system markedly decreases the ability to translate natural mRNA but not poly(U). Compared to extracts from unheated spheroplasts, the following activities in ts 187 extracts from spheroplasts preincubated at 39 degrees C are not significantly affected: activation of methionine and methionylation of tRNAMet; formation of (eukaryotic initiation factor 2.Met-tRNAf.GTP] ternary complex; binding of mRNA to 40S preinitiation intermediate containing Met-tRNAf, and joining of 60S subunits to form the 80S initiation complex; elongation factor 1- and elongation factor 2-dependent elongations reactions; and termination and release of completed polypeptide chains. However, the interaction between the [eukaryotic initiation factor 2.Met-tRNAf.GTP] ternary complex and 40 S subunits, to form the 40 S preinitiation complex, is drastically inhibited by treatment of the spheroplasts at 39 degrees C.

We report the detection in vivo of precursors to the A and the B subunits of the heat-labile enterotoxin (LT) in Escherichia coli. Both pre-LT A (Mr = 29,500) and pre-LT B (Mr = 13,500) are present in the spheroplast fraction of the bacteria after separation of the cells in spheroplasts and periplasm. Two smaller LT A related polypeptides (17 and 23 kDa) were also detected in the spheroplast fraction. Both were degraded with a half-time of about 40 s. Mature subunits (Mr = 27,500 for LT A, and 11,500 for LT B) are released from the spheroplasts soon after processing and occur freely in the periplasm not associated with the cytoplasmic or the outer membranes. Processing occurs mainly post-translationally for both the A and the B subunits. However, they show different kinetics of processing and subsequent segregation into the periplasm. Whereas pre-LT B is processed and released within seconds after chain termination, pre-LT A is processed and released more slowly, and a subfraction of mature LT A may reside in the cytoplasmic membrane for several minutes.

Halobacterium halobium spheroplasts synthesize and accumulate a bacteriorhodopsin precursor. By labeling of the precursor with [35S]Met and [3H]Leu followed by Edman degradation, we have confirmed the previous conclusion from the DNA sequencing that the precursor contains 13 additional amino acids at the NH2 terminus of bacteriorhodopsin. Although not processed in the spheroplasts, it integrates into the purple membrane in the correctly folded conformation. This was shown by the mode of cleavage by a number of proteolytic enzymes, the site of attachment of retinal, and the formation of oligomers on reaction with bifunctional cross-linking reagents. In all these respects, the behavior of the precursor was identical with that of native mature bacteriorhodopsin in the purple membrane. Finally, the precursor was not processed to bacteriorhodopsin even when the spheroplasts were subsequently allowed to revert to rod-shaped cells. This suggests that either the processing of the precursor is cotranslational or that the NH2 terminus of the precursor becomes inaccessible to the processing enzyme in the spheroplasts following integration into the membrane.

The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport. Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe. Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA.

Anaerobic lactose and/or amino acid transport by membrane vesicles prepared from Escherichia coli ML 308-225 can be coupled to at least four electron transfer systems: alpha-glycerol-P-dehydrogenase:nitrate reductase, formate dehydrogenase:nitrate reductase, alpha-glycerol-P dehydrogenase:fumarate reductase, and formate dehydrogenase:fumarate reductase. Vesicles contain one or more of these electron transfer systems depending on the growth conditions of the parent cells. alpha-Glycerol-P dehydrogenase and fumarate reductase are present only in vesicles prepared from cells grown in the presence of glycerol or fumarate, respectively. Formate dehydrogenase and nitrate reductase activities, on the other hand, are present in vesicles from cells grown on a variety of media. alpha-Glycerol-P and formate are able to drive aerobic transport in vesicles prepared from anaerobically grown cells, indicating coupling between aerobic and anaerobic electron transfer systems.

mRNAs of the yeast, Saccharomyces cerevisiae, have modified 5' termini similar but not identical with those present in the higher eukaryotes. Poly(A)-containing RNA isolated from spheroplasts or from polysomes of cells labeled with [methyl-3H]methoionine or 32Pi contain methylated, blocked 5'-terminal structures of composition: m7G(5')pppAp and m7G(5')pppGp, occurring in a relative distribution of 75% and 25%, respectively. Furthermore there is no N6-methyladenosine nor any other methylated nucleoside in the entire molecule of poly(A)-containing RNA. Experiments with a mutant, temperature-sensitive for the transport of mRNA to cytoplasm, suggest that the methylation and addition of the blocking group take place in the nucleus and that the genetic lesion in the mutant does not affect these mechanisms.

The effect of mitomycin C on the synthesis of recA protein in Escherichia coli has been analyzed in a variety of conditions, using an immunoradiometric assay (Paoletti, C., Salles, B., and Giacomoni, P. U. (1982) Biochimie 64, 239-246). In exponentially growing cultures of E. coli AB 1157, the addition of mitomycin C (5 micrograms/ml) promotes a 15-fold increase of the content of recA protein with respect to the basal level. Kinetic analysis of this induction shows that the maximum is reached 60 to 90 min after the addition of the drug and then the level decreases. In an uvrA mutant treated with mitomycin C the level of recA protein reaches a maximum within an hour and afterwards it does not decrease. Treatment of exponentially growing cells with EDTA and lysozyme induces a 3-fold increase of recA protein content, in comparison to the basal level. When such spheroplasts are added with mitomycin or nalidixic acid, a striking increase of the recA protein content in the spheroplast suspension is observed, which tends to level off an hour and a half after the addition of the drugs. The maximum level of recA protein content is five times the level measured after lysozyme treatment, i.e. 15 times the basal level in exponentially growing cells.

We present here a detailed analysis of the effect of amino acid starvation and the addition of cycloheximide on RNA metabolism of yeast cells and spheroplasts. These effects have been studied at the level of uridine phosphorylation, methylation of rRNA, and biosynthesis of 35 S, 4 S, and 5 S RNA species. Amino acid starvation inhibits the phosphorylation of uridine assigned for RNA synthesis more than that for other metabolic processes. This implies that a salvage pathway for the synthesis of UMP and CMP is regulated by the rate of transcription and perhaps is localized in the nucleus. The rate of rRNA methylation is not coupled with the rate of transcription; therefore, quantitation of 35 S RNA synthesis (yeast rRNA primary transcript by [methyl-3H]methionine labeling is unreliable. Biosynthesis of 35 S RNA ceases immediately after the cells are transferred to an amino-acid-deficient medium; at a later time 4 S and 5 S RNAs are also inhibited. Therefore, coordination and noncoordination in the stringent response of these RNA species depend upon the time of starvation. Although addition of a small dose (less than 1.0 microgram/ml) of cycloheximide to starved yeast spheroplasts does not alter the rate of uridine phosphorylation, it increases the rate of entrance of uridine into total RNA. This effect is of greater magnitude in 4 S and 5 S than in 35 S RNA. Since the drug does not alter the rate of decay of 35 S RNA that takes place in starvation, it has a selective effect on transcription. A similar small dose, however, produces inhibition of transcription of all these RNA species in nonstarved conditions. This opposite effect of the drug appears to be a characteristic feature of RNA metabolism in eukaryotes.

A cell-free protein-synthesizing system has been prepared from Saccharomyces cerevisiae by differential centrifugation of lysed spheroplasts. The preparation, a modified 100,000 x g supernatant fraction, contains ribosomes and monosomes, ribosomal subunits, translation factors, and aminoacyl-tRNA synthetases, but no polysomes. After removal of small amounts of remaining mRNA with micrococcal nuclease, protein synthesis is stringently dependent on the addition of mRNA, as well as amino acids and an energy-generating system. The 5'-cap analogue, 7-methylguanosine 5'-phosphate, inhibits translation of several natural mRNAs, but has no effect on chain elongation. Incubation of the polysome-free extract with natural mRNA leads to the formation of protein-synthesizing polysomes and eventually, to the release of protein; the molecular weight of the protein synthesized in the presence of BMV (brome mosaic virus) RNA is consistent with that of BMV coat protein.

The TrkA protein, which is essential for the activity of the constitutive Trk K+-uptake system of Escherichia coli, is a peripheral membrane protein. The protein was detected in immunoblots by polyclonal antibodies to sodium dodecyl sulfate-denatured TrkA protein. In extracts from wild-type cells equal amounts of TrkA were found in the membrane and soluble fractions, suggesting that membrane binding is relatively weak. When the protein was moderately overproduced it appeared mainly in the soluble fraction; stronger overproduction led to the formation of aggregates that could not be solubilized by nonionic detergents. Mutations in the three other genes implicated in Trk activity, trkE, trkG, and trkH, reduced or abolished the binding of TrkA to the membrane. These results support the model, previously based solely on genetic data, that Trk is a multisubunit complex and implicates the products of the other trk genes in the normal binding of TrkA to the complex in the cytoplasmic membrane.

In the yeast Saccharomyces cerevisiae we have carried out a detailed study of the response of transcription to the inhibition of translation. Measurement of the incorporation of labeled bases and nucleosides into the nucleoside triphosphate pools revealed that amino acid deprivation brings about a 10- to 50-fold inhibition of such labeling. Therefore accurate comparisons of RNA synthesis using such precursors are difficult to obtain. To overcome this problem we have turned to the use of L-[methyl-3H] methionine as a precursor, because the labeling of the S-adenosylmethionine pool is relatively unaffected by the rate of protein synthesis. Using this precursor, we have observed that in the absence of protein synthesis the transcription of ribosomal RNA is reduced by 80%, the transcription of messenger RNA is reduced by about 25%, and the transcription of transfer RNA is reduced by less than 20%. These results are obtained when protein synthesis is inhibited either by deprivation of an amino acid or by the addition of cycloheximide. Ribosomal precursor RNA synthesized in the absence of protein synthesis is fully methylated. We conclude that the transcription of rRNA is the primary target of stringent control. Furthermore the inhibition of protein synthesis, itself, may be the trigger for this response.

We report the purification and localization of the fadL gene product (FLP), an essential component of the long-chain fatty acid transport machinery in Escherichia coli. FLP was extracted from total membranes by differential extraction with the nonionic detergents Tween 20 and Triton X-100. This protein was further purified from a Tween 20-insoluble-Triton X-100-soluble extract by salt fractionation, gel filtration chromatography, and hydrophobic interaction chromatography. This regime results in a 95-fold purification of FLP from total membranes. The purified protein preparation was homogeneous based on silver staining and gave the characteristic behavior established for the fadL gene product in the presence of sodium dodecyl sulfate at different temperatures prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr of 33,000 when heated at 25 degrees C and Mr of 43,000 when heated at 100 degrees C) and on two-dimensional polyacrylamide gels (pI of 4.6 and a Mr of 33,000). Purified FLP was rich in hydrophobic residues accounting for approximately 45% of the total amino acid composition. To localize FLP, antisera were raised against the purified protein and were used to probe differentially fractionated membranes by Western immunoblotting. This procedure demonstrated the presence of this protein only in the outer membrane fraction of fadL+ strains. We confirmed the outer membrane localization of FLP by measuring long-chain fatty acid transport in fadL+ and fadL strains treated with EDTA to alter outer membrane permeability and in spheroplasts generated from fadL+ and fadL strains. Both EDTA-treated cells and spheroplasts transported long-chain fatty acids at essentially the same rate regardless of whether they contained a wild-type or mutant fadL gene. These data imply that FLP is a protein in the outer membrane which is specifically involved in long-chain fatty acid transport.

Several methods were used to study the source of energy in the uptake of hexose phosphates by Escherichia coli K12. The uptake was sensitive to inhibition by agents that affect electron transport, such as lack of oxygen, cyanide, and heptylhydroxyquinoline-N-oxide, and by agents that affect ATP utilization, such as dicyclohexylcarbodiimide and arsenate. It was also sensitive to uncouplers in the presence of absence of oxygen. The strain of E. coli used extruded protons during respiration. Uncer anaerobic conditions, the uptake of approximately 1 eg to H+ per glucose 6-phosphate. These observations are consistent with a chemiosmotic mechanism of genergized glucose 6-phosphate uptake. The rate of glucose 6-phosphate uptake was maximal in KC1, but was also stimulated by MgC12 or CaC12. Inhibition by A217, a nigericin-like antibiotic, was prevented by K+ whereas valinomycin and gramicidin inhibited in the presence or absence of K+.

A variety of commercially available cell wall hydrolytic enzyme preparations were screened alone and in various combinations for their ability to degrade the cell wall of Neurospora crassa wild type strain 1A. A combination was found which causes complete conversion of the normally filamentous germinated conidia to spherical structures in about 1.5 h. Examination of these spheroplasts by scanning electron microscopy indicated that, although they are spherical, they retain a smooth coat that can only be removed upon prolonged incubation in the enzyme mixture (about 10 h). The 10-h incubation in the enzyme mixture appears to have no obvious detrimental effects on the integrity of the plasma membrane since the activity and regulatory properties of the glucose active transport system in 10-h spheroplasts are essentially unimpaired. Importantly, plasma membranes can be isolated from the 10-h spheroplasts by an adaptation of the concanavalin A method developed previously in this laboratory for cells of the cell wall-less sl strain, which is not the case for the 1.5-h spheroplasts. The yield of plasma membrane vesicles isolated by this procedure is 18-36% as indicated by surface labeling with diazotized [125I]iodosulfanilic acid, and the preparation is less than 1% contaminated with mitochondrial protein. The chemical composition of the wild type plasma membranes is similar to that previously reported for membranes of the sl strain of Neurospora. The isolated wild type plasma membrane vesicles also exhibit all of the functional properties that have previously been demonstrated for the sl plasma membrane vesicles. The wild type vesicles catalyze MgATP-dependent electrogenic proton translocation as indicated by the concentrative uptake of [14C]SCN- and [14C]imidazole under the appropriate conditions, which indicates that they contain the plasma membrane H+-ATPase previously shown to exist in the sl plasma membranes and that they possess permeability barrier function as well. The vesicles also contain a Ca2+/H+ antiporter as evidenced by their ability to catalyze protonophore-inhibited MgATP-dependent 45Ca2+ accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of the isolated vesicles indicate that the protein composition of the wild type vesicles is roughly similar to that of the sl plasma membranes with the H+-ATPase present as a major band of Mr approximately 105,000. The wild type plasma membrane ATPase forms a phosphorylated intermediate similar to that of the sl ATPase, and the specific activity of the H+-ATPase in both wild type and sl membranes is approximately 3 mumol of Pi released/mg of protein/min.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells of Escherichia coli selectively degrade proteins that have incorporated amino acid analogs. Within 1 hour after exposure of cells to canavanine, 50% of the analog-containing proteins were degraded to acid-soluble form. At the same time, no net loss of canavanine-containing protein occurred from the 100,000 X g supernatant. Instead, most of the proteins containing the analog, unlike normal ones, accumulated in particulate fractions sedimenting at 10,000 X g or 100,000 X g. They were then lost from these fractions concomitant with the degradation of the abnormal proteins. The loss of such proteins from particulate fractions accounted for all of the protein degraded to acid-soluble form. Similar observations were obtained after incorporation of other analogs or puromycin. The 10,000 X g pellets correspond to amorphous dense intracellular granules visible in electron micrographs of cells exposed to canavanine. Upon removal of the analog, these granules disappeared, simultaneously with the degradation of the analog-containing proteins. These pellets do not resemble a degradative organelle, like the lysosome; they are not osmotically sensitive, do not exclude inulin, are not enclosed by a membrane, and do not show autolytic activity. The proteins in the granules could be solubilized by sodium dodecyl sulfate but not by Triton, NaC1, dithiothreitol, RNase, DNase, or phospholipase. The proteins extracted from the pellet with sodium dodecyl sulfate tend to become particulate again upon removal of this detergent. Incorporation of canavanine caused a normally soluble polypeptide, the monomer of beta-galactosidase, to be inactive and found in the sedimentable fraction. These findings suggest that (a) the presence of amino acid analogs in proteins can make them less soluble, and (b) the inclusions are formed by the spontaneous precipitation of abnormal proteins rather than by an active granule-forming process.

Reinfection of Escherichia coli with the bacterial virus T4 causes modifications of the properties of the host cell envelope during the preeplicative phase of the lytic cycle. These changes include altered densities cell enveloped and their subfractions, morphological modifications of membrane vesicles, and association of newly synthesized proteins with the host cell envelope. Polypeptide analysis by high resolution electrophoresis on polyacrylamide slab gels in dodecyl sulfate revealed that most of some 30 prereplicative phage-coded polypeptides are attached to this structure. Different means of cell disruption and selective extraction procedures, such as variations of ionic strength, removal of divalent cations, and the addition of chaotropic agents or detergents were used to study the characteristics of these attachments. Many proteins appeared to be artifactually absorbed or weakly bound to the envelope, Separation of cell walls from plasma membranes showed that all of the tightly bound proteins were associated withthe cell membrane fraction. The partitioning of phage proteins between the different fractions was monitored using 12 polypeptides which were identified as products of distinct phage genes. Of these, 8 were eliminated as potential membrane markers. Four polypeptides, the products of genes rIIA, rIIB, 39, and 52 were operationally defined as membrane proteins. The number of molecules of the 12 identified phage gene products, synthesized during a single lytic cycle, was determined. The results allowed the estimation of the concentration in the membrane of those proteins which were found to be quantitatively associated with that structure. Association of phage proteins with the cell envelope was found to be unaffected by mutations in any of the identified phage polypeptides.

The active transport of maltose in Escherichia coli requires the products of five genes. These include a water-soluble periplasmic maltose-binding protein, three cytoplasmic membrane proteins, and an outer membrane protein. In order to evaluate the role of the maltose-binding protein in active transport, a nonpolar internal deletion of the structural gene for the maltose-binding protein was constructed. A strain which contains this deletion is unable to grow on maltose at an external concentration of 25 mM, even when the remaining components of the transport system are synthesized constitutively. This demonstrates that the periplasmic maltose-binding protein is essential for detectable translocation of maltose across the cytoplasmic membrane. Mal+ revertants of the deletion strain were obtained. In one of these strains, the remaining components of the maltose transport system gained the ability to translocate maltose across the membrane independently of the periplasmic binding protein. Maltose transport in this revertant strain is specific for maltose; it is not inhibited by other alpha and beta glucosides and galactosides. In contrast to the wild type, transport activity in the Mal+ revertant strain is retained by spheroplasts. The cytoplasmic membrane components of the maltose transport system in the revertant appear to form a substrate recognition site. It is likely that this site exists in wild type cells but is available only to substrate molecules that are bound to the maltose-binding protein. A model for the operation of the transport system is presented. In this model, the substrate recognition site in the cytoplasmic membrane is exposed to alternate sides of the membrane.

A procedure has been designed for the simultaneous isolation, in a single step, of the nuclei and cytoplasm of Saccharomyces cerevisiae alphas288c spheroplasts. We have examined the polyadenylate poly(A)-containing RNA in these fractions and their kinetics of synthesis. Nuclear RNA saturates with [3H] adenine within 10 min. Labeled RNA appears very quickly in the cytoplasm, exceeding the amount of labeled nuclear RNA within 2 min after the addition of [3H] adenine. Nuclear poly(A)-containing RNA is approximately 10% of the total cellular poly(A)-containing RNA obtained from spheroplasts labeled for 15 min. Nuclear poly(A)-containing RNA is not as large as the giant heterogeneous nuclear RNA of animal cells. The distribution of molecular size in nuclear and cytoplasmic populations of poly(A)-containing RNA is very broad with the average size of the nuclear species being moderately larger than the cytoplasmic species. Three distinct size classes of poly(A), with different apparent kinetic properties, are obtained from yeast poly(A)-containing RNA. Their electrophoretic mobility suggests molecular lengths of approximately 20, 40, and 60 nucleotides (Groner, G., Hynes, N., and Phillips, S. (1975) Biochemistry, 13, 5378-5383). Each of these poly(A) classes are present in the mRNA from large and small polyribosomes.

Polysomes consisting of two to eight monosomes were isolated from yeast mitochondria by lysing the mitochondria with Triton X-100 and centrifugation in a 20 to 40% linear sucrose gradient. When yeast spheroplasts were pulse-labeled with [3H]-Leucine in the presence of cycloheximide to block cytoplasmic protein synthesis, radioactivity which was trichloroacetic acid-precipitable was present mainly in the polysome region. Incorporation of leucine was blocked by erythromycin, a specific inhibitor of mitochondrial protein synthesis. Release of radioactivity to the top of the gradient resulted from treating labeled polysomes with either puromycin or ribonuclease (in the latter case with the breakdown of polysomes), indicating that the radioactivity was present in nascent polypeptide chains. Yeast cells were grown in chloramphenicol for 3 hours and in fresh medium for 1 hour and then pulse-labeled with either [3H]leucine or [14C]formate. Three parameters showed a 2-fold increase in cells grown in chloramphenicol prior to pulse labeling: the polysome to monosome ratio, the amount of labeled precursor incorporated into proteins, and the rate of polypeptide chain initiation as judged by the formation of fMet-puromycin. Conversely, these parameters were all decreased approximately 50% in cells treated with cycloheximide prior to pulse labeling. Mitochondria were also isolated from cells previously grown in chloramphenicol or cycloheximide and incubated in vitro with [3H]leucine under optimal conditions. Acid-precipitable radioactivity in the polysome region was increased 3-fold in mitochondria from cells grown previously in chloramphenicol and decreased 75% in those grown in cycloheximide. Furthermore, chain initiation was deomonstrated in the isolated mitochondria by formation of fMet-puromycin. The rate of chain initiation in vitro was increased 2-fold in mitochondria isolated from chloramphenicol-treated cells.