Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase.

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over-production of subunit b of E. coli F(1)F(o) ATP synthase in the mutant host strains E. coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies. Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over-producing subunit b. The new proliferated membranes contained all the over-expressed protein and could be recovered by a single centrifugation step. Recombinant subunit b represented up to 80% of the protein content of the membranes. The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin.

Topology and transport of membrane lipids in bacteria.

The last two decades have witnessed a break-through in identifying and understanding the functions of both the proteins and lipids of bacterial membranes. This development was parallelled by increasing insights into the biogenesis, topology, transport and sorting of membrane proteins. However, progress in research on the membrane distribution and transport of lipids in bacteria has been slow in that period. The development of novel biochemical in vitro approaches and recent genetic studies have increased our understanding of these subjects. The aim of this review is to present an overview of the current knowledge of the distribution and transport of lipids in both Gram-positive and Gram-negative bacteria. Special attention is paid to recently obtained results, which are expected to inspire further research to finally unravel these poorly understood phenomena.

Lipid metabolism and regulation of membrane trafficking.

The past 20 years have witnessed tremendous progress in our understanding of the molecular machinery that controls protein and membrane transport between organelles (Scheckman R, Orci L. Coat proteins and vesicle budding. Science 1996;271: 1526-1533 and Rothman JE. Mechanisms of intracellular protein transport. Nature 1994;372: 55-63.) The research efforts responsible for these impressive advances have largely focused on the identification and characterization of protein factors that participate in membrane trafficking events. The role of membranes and their lipid constituents has received considerably less attention. Indeed, until rather recently, popular models for mechanisms of membrane trafficking had relegated membrane lipids to the status of a passive platform, subject to deformation by the action of coat proteins whose polymerization and depolymerization govern vesicle budding and fusion reactions. The 1990s, and particularly its last half, has brought fundamental reappraisals of the interface of lipids and lipid metabolism in regulating intracellular membrane trafficking events. Some of the emerging themes are reviewed here.

Rapid transmembrane movement of newly synthesized phosphatidylethanolamine across the inner membrane of Escherichia coli.

For the first time the transmembrane movement of an endogenously synthesized phospholipid across the inner membrane of E. coli is reported. [14C]phosphatidylethanolamine (PE) was biosynthetically introduced into inner membrane vesicles from the PE-deficient strain AD93, by reconstitution with the enzyme phosphatidylserine (PS) synthetase. Upon addition of wild type cell lysate containing PS synthetase, and the metabolic substrates CTP and [14C]serine to inside-out vesicles from AD93, [14C]PS was synthesized, which was for the most part converted into [14C]PE. [14C]PE was introduced in right-side out vesicles by enclosing PS synthetase and CTP in the vesicle lumen and adding [14C]serine. The newly synthesized [14C]PE immediately equilibrated over both membrane leaflets (t1/2 less than one min), as determined by its accessibility toward the amino-reactive chemical fluorescamine. In both inside- out and right-side out vesicles, a 35-65% distribution was found of the newly synthesized PE over the cytoplasmic and periplasmic leaflet, respectively. The transport process of PE was not influenced by the presence of ATP or the proton motive force in inside out vesicles. Pretreatment of both types of vesicles with sulfhydryl reagents, or of right-side out vesicles with proteinase K, did not affect the rate and extent of the transmembrane distribution of the newly synthesized PE.

On the accessibility of phosphatidylglycerol to periodate in Escherichia coli.

The transverse distribution of phosphatidylglycerol (PG) across the inner membrane of Escherichia coli was studied. The oxidation of PG by periodate was used to discriminate between the PG present in the cytoplasmic and in the periplasmic leaflet of the inner membrane. Applied to large unilamellar vesicles derived of an E. coli lipid extract, the periodate method demonstrated the expected symmetrical distribution of PG over the bilayer. However, both in right-side-out and in inside-out inner membrane vesicles isolated from E. coli, as well as in intact E. coli cells, the entire pool of PG was oxidized by periodate. The oxidation reaction proceeded at such a high rate that it was impossible to determine the distribution of PG across the membrane. Apparently, periodate easily permeates through the membrane. This permeation could not be inhibited or slowed down, and it was concluded that the use of periodate as oxidizer of PG is not a suitable method to determine the transverse distribution of this phospholipid in the Gram-negative bacterium E. coli. However, periodate can be used to selectively modify the total PG pool in this organism.

Rapid transmembrane movement of C6-NBD-labeled phospholipids across the inner membrane of Escherichia coli.

In this study we have investigated the transmembrane movement of short chain fluorescently labeled phospholipids across the inner membrane of Escherichia coli. Exogenously added C6-NBD-labeled phospholipids rapidly flip across the inner membrane of E. coli, as was shown by a dithionite reduction assay applied to inverted inner membrane vesicles (IIMV) isolated from wild type E. coli cells. The rate of transmembrane movement of the phospholipid probes incorporated into IIMV is temperature dependent, and shows no phospholipid head group specificity. C6-NBD-labeled phospholipids translocate across the membrane of IIMV incubated at 37 degrees C with a t1/2 of 7 min. After the incorporation into IIMV C6-NBD-PG is partially converted to CL by CL-synthase. If IIMV are pretreated with proteinase K the conversion of this fluorescent probe to C6-NBD-CL is not observed anymore, suggesting that the catalytic domain of CL-synthase is at the cytoplasmic site of the plasma membrane of E. coli. Newly synthesized C6-NBD-CL also flips across the inner membrane although at a slower rate than the other phospholipid probes. The transmembrane movement occurs in both directions and is not influenced by treatment of the IIMV with a sulfhydryl reagent or a proteinase, nor by the presence of ATP, or a deltapH across the membrane of the IIMV. However, the transmembrane movement of the C6-NBD-labeled phospholipid probes is not observed in LUVETs (large unilamellar vesicles made by extrusion technique) prepared of wild type E. coli lipids, indicating that the rapid transmembrane movement of phospholipids across the inner membrane of E. coli is a protein-mediated process.

The neuroendocrine protein 7B2 contains unusually potent transcriptional activating sequences.

The expression of the 7B2 protein, secreted from a variety of neural and endocrine tissues, increases dramatically in specific neuroendocrine tumors. We have recently shown that human 7B2 can act as a molecular chaperone in the deaggregation of proteins in vitro. In order to identify polypeptides which might bind 7B2 in vivo, the yeast two-hybrid system was employed. Surprisingly, mere covalent linkage of 7B2 to the DNA-binding domains of two yeast transcription activators, Ace1 and Gal4, activates transcription from the ACE1 and GAL4 operon. 7B2's ability to activate nuclear transcription surpasses that of Ace1 and compares favourably with the strong activation domain of the tumor suppressor protein, p53. Our results suggest that 7B2 must possess an activating sequence, a domain which defines all transcriptional activator proteins. Like the acidic activation domains of some transcriptional activators, 7B2 also binds the yeast TATA-box binding protein, an essential polypeptide in the basic transcription machinery. Deletion analysis of the gene encoding 7B2 reveals two independent transcriptional activating sequences in the 185 amino acid protein. It is therefore conceivable that 7B2 not only has a functional role in the secretory pathway but also in the nucleus. Moreover, these findings raise an intriguing question regarding the activation domains of 7B2 and their possible link to 7B2's oncogenic potential.

The neuroendocrine protein 7B2 acts as a molecular chaperone in the in vitro folding of human insulin-like growth factor-1 secreted from yeast.

The neuroendocrine protein 7B2 prevents premature activation of PC2, an enzyme involved in the processing of prohormones in the secretory pathway. We inquired if this chaperone-like function encompasses a broader role for 7B2 in the folding of hormone-like proteins. As a test, the fate of misfolded human insulin-like growth factor-1 (IGF1) was assessed, in the presence and absence of 7B2. Most of the recombinant IGF1 molecules, secreted from yeast, are a conglomeration of inactive multimers which are either disulfide-linked or mere physical aggregates. We find that yeast-produced 7B2 influences the in vitro conversion of inactive molecules into active monomers. However, the amounts of disulfide-linked dimers remain unaffected during this conversion. Interestingly, both 7B2 and the molecular chaperone DnaK interact with IGF1 in the yeast two-hybrid system. Like DnaK, 7B2 also binds the tumor suppressor protein p53. Binding of DnaK to exposed epitopes of aggregated proteins is known to be a prerequisite for deaggregation. It is conceivable that 7B2 participates in an analogous manner in the dissociation of non-covalently linked multimers of IGF1. Our results indicate that 7B2 might find an application in the deaggregation of potentially useful therapeutic proteins.

Elevated cytosolic concentrations of SecA compensate for a protein translocation defect in Escherichia coli cells with reduced levels of negatively charged phospholipids.

Cellular extracts from cells with reduced synthesis of negatively charged phospholipids were found to support in vitro translocation of the precursor of the outer membrane protein PhoE with increased efficiency. Analysis of these extracts revealed that they contain increased levels of SecA. SecA depletion resulted in a loss of the translocation stimulatory activity, which could be restored by re-addition of purified SecA. We conclude that elevated cytosolic levels of SecA counteract the reduction of translocation efficiency due to low levels of negatively charged phospholipids in the inner membrane.