Rosetta N. Reusch

Poly-3-hydroxybutyrate/polyphosphate complexes form voltage-activated Ca2+ channels in the plasma membranes of Escherichia coli

The lipidic polymer, poly-3-hydroxybutyrate (PHB), is found in the plasma membranes of Escherichia col complexed to calcium polyphosphate (CaPPi). The composition, location, and putative structure of the polymer salt complexes led Reusch and Sadoff (1988) to propose that the complexes function as Ca2+ channels. Here we use bilayer patch-clamp techniques to demonstrate that voltage-activated Ca2+ channels composed of PHB and CaPPi are in the plasma membranes of E. coli. Single channel calcium currents were observed in vesicles of plasma membranes incorporated into planar bilayers of synthetic 1-palmitoyl, 2-oleoyl phosphatidylcholine. The channels were extracted from cells and incorporated into bilayers, where they displayed many of the signal characteristics of protein Ca2+ channels: voltage-activated selective for divalent over monovalent cations, permeant to Ca2+, manner by La3+, Co2+, Cd2+, and Mg2+, in that order. The channel-active extract, purified by size exclusion chromatography, was found to contain only PHB and CaPPi. This composition was confirmed by the observation of comparable single channel currents with complexes reconstituted from synthetic CaPPi and PHB, isolated from E. coli. This is the first report of a biological non-proteinaceous calcium channel. We suggest that poly-3-hydroxybutyrate/calcium polyphosphate complexes are evolutionary antecedents of protein Ca2+ channels.

Poly-β-hydroxybutyrate/Calcium Polyphosphate Complexes in Eukaryotic Membranes

Abstract Poly-β-hydroxybutyrate/calcium polyphosphate (PHB-CaPolyPi) complexes exist as labile quasi-crystalline structures in bacterial plasma membranes. The composition, structure, and distribution of the complex suggest it may play a role in the regulation of intracellular calcium and in calcium signaling. The importance of these functions led to this investigation of the occurrence of PHB-CaPolyPi complexes in eukaryotes. A variety of plant and animal systems were analyzed and all were found to contain PHB associated with CaPolyPi. The intracellular location of the complex in bovine liver was primarily the mitochondria and microsomes, with smaller amounts in the plasma membranes. Eukaryotic PHB had the same narrow range of chain lengths (120–200 subunits) as PHB in bacterial membranes, and was associated with PolyPi of somewhat greater length (170–220) than the bacterial counterpart (130–170).

Novel components and enzymatic activities of the human erythrocyte plasma membrane calcium pump

The plasma membrane Ca2+ pump is essential for the maintenance of cystolic calcium ion concentration levels in eukaryotes. Here we show that the Ca2+‐ATPase, purified from human erythrocytes, contains two homopolymers, poly(3‐hydroxybutyrate) (PHB) and inorganic polyphosphate (polyP), which form voltage‐activated calcium channels in the plasma membranes of Escherichia coli and other bacteria. Furthermore, we demonstrate that the plasma membrane Ca2+‐ATPase may function as a polyphosphate kinase, i.e. it exhibits ATP‐polyphosphate transferase and polyphosphate‐ADP transferase activities. These findings suggest a novel supramolecular structure for the functional Ca2+‐ATPase, and a new mechanism of uphill Ca2+ extrusion coupled to ATP hydrolysis.

Posttranslational modification of E. coli histone-like protein H-NS and bovine histones by short-chain poly-(R)-3-hydroxybutyrate (cPHB)

Short‐chain poly‐(R)‐3‐hydroxybutyrate (cPHB), a highly flexible, amphiphilic molecule with salt‐solvating properties, is a ubiquitous constituent of prokaryotic and eukaryotic cells, wherein it is mainly conjugated to proteins. The solvating properties and cellular distribution of cPHB suggest it may be associated with proteins that bind and/or transfer DNA. Here we examine Escherichia coli protein H‐NS and calf thymus histones, H1, H2A, H2B, H3, and H4, for the presence of cPHB. The proteins are related in that all bind to DNA and are implicated in the compact organization of the chromosome. The presence of cPHB in E. coli H‐NS was first detected in Western blots of two‐dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels of total cell proteins, probed with anti‐cPHB IgG, and then by Western blot analysis of the purified protein. Western blot analysis of the calf thymus histones indicated that each contained cPHB. The presence of cPHB in H‐NS and histones was confirmed by chemical assay. The in vivo size of conjugated cPHB could not be established due to the lack of standards and degradation of cPHB during protein purification and storage. The molecular characteristics of cPHB and its presence in histone‐like and histone proteins of diverse organisms suggest it may play a role in DNA binding and/or DNA organization.

Outer membrane protein A of Escherichia coli forms temperature-sensitive channels in planar lipid bilayers

The temperature dependence of single‐channel conductance and open probability for outer membrane protein A (OmpA) of Escherichia coli were examined in planar lipid bilayers. OmpA formed two interconvertible conductance states, small channels, 36–140 pS, between 15 and 37°C, and large channels, 115–373 pS, between 21 and 39°C. Increasing temperatures had strong effects on open probabilities and on the ratio of large to small channels, particularly between 22 and 34°C, which effected sharp increases in average conductance. The data infer that OmpA is a flexible temperature‐sensitive protein that exists as a small pore structure at lower temperatures, but refolds into a large pore at higher temperatures.

Insights into the structure and assembly of Escherichia coli outer membrane protein A

Outer membrane protein A (OmpA) of Escherichia coli is a paradigm for the biogenesis of outer membrane proteins; however, the structure and assembly of OmpA have remained controversial. A review of studies to date supports the hypothesis that native OmpA is a single‐domain large pore, while a two‐domain narrow‐pore structure is a folding intermediate or minor conformer. The in vitro refolding of OmpA to the large‐pore conformation requires isolation of the protein from outer membranes with retention of an intact disulfide bond followed by sufficient incubation in lipids at temperatures of ≥ 26 °C to overcome the high energy of activation for refolding. The in vivo maturation of the protein involves covalent modification of serines in the eighth β‐barrel of the N‐terminal domain by oligo‐(R)‐3‐hydroxybutyrates as the protein is escorted across the cytoplasm by SecB for post‐translational secretion across the secretory translocase in the inner membrane. After cleavage of the signal sequence, protein chaperones, such as Skp, DegP and SurA, guide OmpA across the periplasm to the β‐barrel assembly machinery (BAM) complex in the outer membrane. During this passage, a disulfide bond is formed between C290 and C302 by DsbA, and the hydrophobicity of segments of the C‐terminal domain, which are destined for incorporation as β‐barrels in the outer membrane bilayer, is increased by covalent attachment of oligo‐(R)‐3‐hydroxybutyrates. With the aid of the BAM complex, OmpA is then assembled into the outer membrane as a single‐domain large pore.

Physiological importance of poly-(R)-3-hydroxybutyrates.

Poly‐(R)‐3‐hydroxybutyrates (PHB), linear polymers of (R)‐3‐hydroxybutyrate, are components of all biological cells in which short polymers (<200 monomer residues) are covalently attached to certain proteins and/or noncovalently associated with polyphosphates – inorganic polyphosphate (polyP), RNA, and DNA. The low concentrations, lack of unusual atoms or functional groups, and flexible backbones of this complexed PHB, referred to as cPHB, make them invisible to many analytical procedures; whereas other physical properties – water‐insolubility, high intrinsic viscosity, temperature sensitivity, multiple bonding interactions with other molecules – make them requisite participants in vital physiological processes as well as contributors to the development of certain diseases.

Insight into the selectivity and gating functions of Streptomyces lividans KcsA.

Streptomyces lividans KcsA is a 160-aa polypeptide that oligomerizes to form a tetrameric potassium channel. The three-dimensional structure of the polypeptides has been established, but the selectivity and gating functions of the channel remain unclear. It has been shown that the polypeptides copurify with two homopolymers, poly[(R)-3-hydroxybutyrate] (PHB) and inorganic polyphosphate (polyP), which have intrinsic capacities for cation selection and transport. PHB/polyP complexes are highly selective for divalent cations when pH is greater than the pK2 of polyP (≈6.8), but this preference is lost when pH is ≤pK2. It is postulated that KcsA polypeptides attenuate the divalent negative charge of the polyP end unit at physiological pH by strategic positioning of two C-terminal arginines. Here we mutate one or both of the C-terminal arginines and observe the effects on channel selectivity in planar lipid bilayers. We find that channels formed by KcsA polypeptides that retain a single C-terminal arginine remain highly selective for K+ over Mg2+, independent of medium pH; however, channels formed by KcsA polypeptides in which both C-terminal arginines have been replaced with neutral residues are selective for Mg2+ when pH is >7 and for K+ when pH is <7. Channel gating may be triggered by changes in the balance between the K+ polyP− binding energy, the membrane potential, and the gradient force. The results reveal the importance of the C-terminal arginines to K+ selectivity and argue for a supramolecular structure for KcsA in which the host polypeptides modify the cation preference of a guest PHB/polyP complex.

Poly-3-hydroxybutyrate synthase from the periplasm of Escherichia coli.

This is the first report of a poly-3-hydroxybutyrate (PHB) synthase in Escherichia coli. The enzyme was isolated from the periplasm using ammonium sulfate fractionation, hydrophobic, and size-exclusion chromatography and identified by LC/MS/MS as YdcS, a component of a putative ABC transporter. Green Fluorescent Protein-tagged ydcS, purified by 2D native gel electrophoresis, also exhibited PHB synthase activity. Optimal conditions for enzyme activity were 37 degrees C, pH 6.8-7.5, 100 mM KCl. K(m) was 0.14 mM and V(max) was 18.7 nmol/mg protein/min. The periplasms of deletion mutants displayed <25% of the activity of the parent strain. Deletion mutants exhibited approximately 25% less growth in M9 medium, glucose, and contained approximately 30% less PHB complexed to proteins (cPHB) in the outer membranes, but the same concentration of chloroform-extractable PHB as wild-type cells. The primary sequence of YdcS suggests it may belong to the alpha-/beta-hydrolase superfamily which includes polyhydroxybutyrate (PHB) synthases, lipases, and esterases.

A mechanism for phosphoglyceride and Ca2+ transbilayer movement

The ionophoretic capabilities of phosphoglycerides (PL) have been examined by measuring their translocation via cations from aqueous dispersions into linear and cyclic hydrocarbons. The PL surveyed were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI). Only PA displayed ionophoretic activity in single lipid dispersions with a cation selectivity order of Mn greater than Ca. PG, PE and PC, but not PI, had a synergistic affect of PA induced translocation. These PL, inactive individually or in any combination, became strong Ca2+ ionophores of variable activity in association with PA. A dimeric structure proposed for the ionophoretic species forms the basis of a mechanism for transbilayer movement of PA, PG, PE and PC which would establish an asymmetric distribution of these lipids in the two faces of the bilayer by equilibrium processes.