Sequences determining the cytoplasmic localization of a chemoreceptor domain.

The Escherichia coli serine chemoreceptor (Tsr) is a protein with a simple topology consisting of two membrane-spanning sequences (TM1 and TM2) separating a large periplasmic domain from N-terminal and C-terminal cytoplasmic regions. We analyzed the contributions of several sequence elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions. The principal findings were as follows. (i) The cytoplasmic localization of the C-terminal domain depended on TM2 but was quite tolerant of mutations partially deleting or introducing charged residues into the sequence. (ii) The basal level of C-terminal domain export was significantly higher in proteins with the wild-type periplasmic domain than in derivatives with a shortened periplasmic domain, suggesting that the large size of the wild-type domain promotes partial membrane misinsertion. (iii) The membrane insertion of deletion derivatives with a single spanning segment (TM1 or TM2) could be controlled by either an adjacent positively charged sequence or an adjacent amphipathic sequence. The results provide evidence that the generation of the Tsr membrane topology is an overdetermined process directed by an interplay of sequences promoting and opposing establishment of the normal structure.     

Secondary structure of a KCNE cytoplasmic domain

Type I transmembrane KCNE peptides contain a conserved C-terminal cytoplasmic domain that abuts the transmembrane segment. In KCNE1, this region is required for modulation of KCNQ1 K(+) channels to afford the slowly activating cardiac I(Ks) current. We utilized alanine/leucine scanning to determine whether this region possesses any secondary structure and to identify the KCNE1 residues that face the KCNQ1 channel complex. Helical periodicity analysis of the mutation-induced perturbations in voltage activation and deactivation kinetics of KCNQ1-KCNE1 complexes defined that the KCNE1 C terminus is alpha-helical when split in half at a conserved proline residue. This helical rendering assigns all known long QT mutations in the KCNE1 C-terminal domain as protein facing. The identification of a secondary structure within the KCNE1 C-terminal domain provides a structural scaffold to map protein-protein interactions with the pore-forming KCNQ1 subunit as well as the cytoplasmic regulatory proteins anchored to KCNQ1-KCNE complexes.     

Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein

The large majority of histidine kinases (HKs) are multifunctional enzymes having autokinase, phosphotransfer and phosphatase activities, and most of these are transmembrane sensor proteins. Sensor HKs possess conserved cytoplasmic phosphorylation and ATP-binding kinase domains. The different enzymatic activities require participation by one or both of these domains, implying the need for different conformational states. The catalytic domains are linked to the membrane through a coiled-coil segment that sometimes includes other domains. We describe here the first crystal structure of the complete cytoplasmic region of a sensor HK, one from the thermophile Thermotoga maritima in complex with ADPbetaN at 1.9 A resolution. The structure reveals previously unidentified functions for several conserved residues and reveals the relative disposition of domains in a state seemingly poised for phosphotransfer. The structure thereby inspires hypotheses for the mechanisms of autophosphorylation, phosphotransfer and response-regulator dephosphorylation, and for signal transduction through the coiled-coil segment. Mutational tests support the functional relevance of interdomain contacts.     

Glutathione: a key component of the cytoplasmic labile iron pool

The cytoplasmic labile iron pool supplies iron to the mitochondrion for heme and iron sulfur cluster synthesis and to many cytoplasmic enzymes, thereby controlling numerous metabolic reactions. Surprisingly the chemical nature of this pool has never been convincingly characterised. Here we provide evidence for iron(II)glutathione being the dominant component of this pool. We report for the first time the affinity constant for the glutathione–iron(II) interaction and use this value to study the cytoplasmic speciation of iron(II). The formation of this complex is a major determinant of the electrode potential of the cytoplasmic ferrous iron pool, a means of selecting between iron(II) and manganese(II) and it provides a substrate for glutaredoxin/iron clusters at the dimer interface of glutaredoxins involved in the synthesis of Fe–S cluster proteins.     

Structural insights into the cytoplasmic domain of a human BK channel

The large conductance, voltage- and Ca(2+) -activated K(+) (BK or Slo1) channel is widely expressed in mammalian cells/tissues (i.e. neurons, skeletal and smooth muscles, exocrine cells, the inner ear) and regulates action potential firing, muscle contraction and secretion. The large ionic conductance and unusual, dual stimulus-driven gating behavior of this channel have long intrigued membrane biophysicists, and recent structure/function analyses have provided increasingly detailed insights into the molecular "bells and whistles" that regulate BK channel activity. Now, in two complementary articles published by the groups of Rod MacKinnon and Youxing Jiang, high resolution x-ray crystal structures of the human BK channel's large cytoplasmic domain have been solved in both the absence and presence of bound Ca(2+), conditions which would presumably promote the resting and activated conformations of this large domain. Given the regulatory importance of the cytosolic domain on BK channel gating, these experimentally determined structures reveal a number of key insights, including: 1) the physical arrangement and interactions of the tandem RCK1 and RCK2 domains within a single channel subunit, 2) the assembly of the four large cytoplasmic domains into a symmetric, tetrameric complex, 3) the formation of the channel's "gating ring" structure, based on the assembly of the individual RCK1 and 2 domains, and 4) the structural elements underlying the regions critical for divalent metal ion binding (i.e. Ca (2+) and Mg (2+)) and their potential influence on conduction pore.     

Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein

The previously uncharacterized third and fourth genes (pulE and pulF) of the pullulanase secretion gene operon of Klebsiella oxytoca strain UNF5023 are, respectively, predicted to encode a 55 kDa polypeptide with a putative nucleotide-binding site, and a highly hydrophobic 44 kDa polypeptide that probably spans the cytoplasmic membrane several times. Expression of pulE in minicells or under the control of a strong bacteriophage T7 promoter resulted in the production of a c. 58 kDa cytoplasmic protein. A representative PulE-beta-galactosidase hybrid protein created by Tnlac mutagenesis was also found mainly in the cytoplasm. These results are in line with the predicted absence from PulE of a region of sufficient hydrophobicity to function as a signal sequence. The PulF polypeptide could not be detected either in minicells or when the gene was transcribed from the T7 promoter, but the acquirement of three pulF-lacZ gene fusions that encoded hybrid proteins with relatively high levels of beta-galactosidase activity indicates that this gene can be transcribed and translated. Gene disruption experiments indicated that both pulE and pulF are required for pullulanase secretion in Escherichia coli K-12. Both proteins exhibit considerable homology throughout their entire lengths with other proteins involved in protein secretion, pilin assembly, conjugation and transformation competence in a variety of bacteria. In addition, PulE protein has consensus sequences found in a wide variety of nucleotide-binding proteins. This study completes the initial characterization of the pullulanase secretion gene operon, which comprises 13 genes that are all essential for the transport of pullulanase across the outer membrane.     

Further observations on a cytoplasmic structure in the hamster oocyte

Summary Cytoplasmic structures consisting of two loops of “unit membrane” connected by a region of straight or curved 7-layered membrane are described. It is suggested that this appearance is caused by the collapse of a vesicle lined with a thin coating of electron-dense material.     

EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli

Abstract A gene product with an apparent molecular mass of approximately 39000 Da can be identified in the cytoplasmic membrane of Escherichia coli upon expression of cloned envC . In this communication we report that the product was labelled with [³H]glycerol and [³H]palmitic acid, and a precursor molecule of increased molecular mass was accumulated when cells were treated with globomycin, a specific inhibitor for the prolipoprotein signal peptidase. The same precursor molecule was encoded by an envC mutant gene, in which the cysteine residue in a pentapeptide sequence, Leu-Ile-Ala-Gly-Cys²⁴ within the amino terminal region of EnvC, was replaced by tryptophane (Trp²⁴). This protein was not labelled with [³H]glycerol. The results demonstrate that the envC gene product represents a new lipoprotein of the cytoplasmic membrane of E. coli .     

Functional analysis of a cytoplasmic domain-deleted mutant of the CD4 molecule

The CD4 molecule is a receptor found on a subset of T lymphocytes. It has been proposed that, upon binding MHC class II molecules expressed on APC, the CD4 molecule enhances the responsiveness of the T cell by increasing intercellular avidity and/or by transducing an intracellular signal. We have analyzed the effect of removing the cytoplasmic domain of the CD4 molecule on the ability of the CD4 molecule to enhance T cell responsiveness. The cytoplasmic domain-deleted mutant of the CD4 molecule (CD4 delta) was found to be as efficient as the CD4 molecule at enhancing responsiveness to cells bearing the appropriate Ag. If subcellular Ag in the form of purified Ag incorporated into liposomes was used, the CD4 molecule was found to be much more efficient than the CD4 delta molecule at enhancing responsiveness. However, the defect in the ability of the CD4 delta molecule to enhance responsiveness could be compensated for by increasing the level of expression of the CD4 delta molecule.     

Spermiogenesis in the bluegill (Lepomis macrochirus): a study of cytoplasmic events including cell volume changes and cytoplasmic elimination

The process involved in the reduction of both nuclear and cytoplasmic volume was investigated in the bluegill (Lepomis macrochirus), a teleost fish. Young spermatids contained centrally positioned nuclei which, with time, moved toward the cell surface to become eccentrically positioned. Chromatin condensation was initiated from a region near the implantation fossa, whereas at the opposite pole of the nucleus an area sparse in heterochromatin (clear area) was noted. The nuclear membrane lying adjacent to the clear area dissolved and subsequently reformed, yielding a nucleus with a reduced volume. During this process, packets of cytoplasm surrounded by a double membrane were formed along the future midpiece. The packets of cytoplasm migrated toward the cell surface, protruded from the surface, and were extruded into the spermatocyst lumen. These structures, termed residual bodies, were subsequently endocytosed, accumulated into large phagocytic vacuoles, and eventually degraded by the nearby Sertoli cell. When the spermatocyst ruptured, spermatozoa containing sparse cytoplasm were released into the excurrent duct system. During spermiogenesis, both the nuclear and cytoplasmic volumes decreased substantially (80%, 92% respectively) leading to an overall 87% reduction in total cell volume.     

Spermiogenesis in the bullfrog (Rana catesbeiana): a study of cytoplasmic events including cell volume changes and cytoplasmic elimination

The process by which spermatid cytoplasmic volume is reduced and cytoplasm eliminated during spermiogenesis was investigated in the bullfrog Rana catesbeiana. At early phases of spermiogenesis, newly formed, rounded spermatids were found within spermatocysts. As acrosomal development, nuclear elongation, and chromatin condensation occurred, spermatid nuclei became eccentric within the cell. A cytoplasmic lobe formed from the caudal spermatid head and flagellum and extended toward the seminiferous tubule lumen. The cytoplasmic lobe underwent progressive condensation whereby most of its cytoplasm became extremely electron dense and contrasted sharply with numerous electron-translucent vesicles contained therein. At the completion of spermiogenesis, many spermatids with their highly condensed cytoplasm still attached were released from their Sertoli cell into the lumen of the seminiferous tubule. There was no evidence of the phagocytosis of residual bodies by Sertoli cells. Because spermatozoa are normally retained in the testis in winter and are not released until the following breeding season, sperm were induced to traverse the duct system with a single injection of hCG. Some spermatids remained attached to their cytoplasm during the sojourn through the testicular and kidney ducts; however, by the time the sperm reached the Wolffian duct, separation had occurred. The discarded cytoplasmic lobe (residual body) appeared to be degraded with the epithelium of the Wolffian duct. It was determined that the volume of the spermatid was reduced by 87% during spermiogenesis through a nuclear volume decrease of 76% and cytoplasmic volume decrease of 95.3%.     

Cytogenetics of flower modification of a cytoplasmic male-sterile tobacco

Plants combining the cytoplasm of Nicotiana debneyi and the 48 chromosomes from N. tabacum are male sterile. Early backcross generations of the amphidiploid hybrid to male N. tabacum produced a great variety of plants from which a series of phenotypes with characteristic flower forms and transmission rates have been isolated. Type 1A possesses completely feminized stamens and deeply split corollas, breeds true when backcrossed to normal males and carries 48 N. tabacum chromosomes. Other phenotypes, 2C, 3E and 4H, range toward normal morphology of corollas and stamens. Like 1A, 2C forms no anther tissue and has 48 chromosomes. This type is transmitted to 36.3% of the backcross progeny, the remainder being of type 1A; presumably 2C carries a chromosome segment from N. debneyi that is responsible for the partial restoration of flower structure. In contrast, both 3E and 4H produce anthers and possess an extra chromosome. The extra chromosomes are transmitted to only 19.9% and 7.1% of the progeny, respectively. Significantly, the extra chromosomes found in the anther-forming types are nucleolus organizing and carry a satellite from N. debneyi. On the basis of these observations, we surmise that differentiation of anthers in plants with N. debneyi cytoplasm may depend on the presence of a nucleolus-organizing chromosome from that species. This chromosome is unstable; unaltered, it conditions a highly restored phenotype (4H), but when structurally modified, it may control different phenotypic expressions. Other examples of satellited restorer chromosomes had been reported for different cytoplasmically male-sterile combinations; therefore, the phenomenon may have more general significance.     

Characterization of a cytoplasmic trehalase of Escherichia coli.

Escherichia coli can synthesize trehalose in response to osmotic stress and is able to utilize trehalose as a carbon source. The pathway of trehalose utilization is different at low and high osmolarity. At high osmolarity, a periplasmic trehalase (TreA) is induced that hydrolyzes trehalose in the periplasm to glucose. Glucose is then taken up by the phosphotransferase system. At low osmolarity, trehalose is taken up by a trehalose-specific enzyme II of the phosphotransferase system as trehalose-6-phosphate and then is hydrolyzed to glucose and glucose-6-phosphate. Here we report a novel cytoplasmic trehalase that hydrolyzes trehalose to glucose. treF, the gene encoding this enzyme, was cloned under ara promoter control. The enzyme (TreF) was purified from extracts of an overexpressing strain and characterized biochemically. It is specific for trehalose exhibiting a Km of 1.9 mM and a Vmax of 54 micromol of trehalose hydrolyzed per min per mg of protein. The enzyme is monomeric, exhibits a broad pH optimum at 6.0, and shows no metal dependency. TreF has a molecular weight of 63,703 (549 amino acids) and is highly homologous to TreA. The nonidentical amino acids of TreF are more polar and more acidic than those of TreA. The expression of treF as studied by the expression of a chromosomal treF-lacZ fusion is weakly induced by high osmolarity of the medium and is partially dependent on RpoS, the stationary-phase sigma factor. Mutants producing 17-fold more TreF than does the wild type were isolated.