The metabolism of lactose by Streptococcus thermophilus is highly regulated, allowing the bacterium to prefer lactose over glucose as main source of carbon and energy. In vitro analysis of the ...enzymes involved in transport and hydrolysis of lactose showed that the transport reaction benefits from the hydrolysis of lactose at the trans side of the membrane. Furthermore, the activity of LacS is modulated by PEP-dependent phosphorylation of the IIA domain via the general energy coupling proteins of the PTS, Enzyme I and HPr. To determine whether unphosphorylated LacS-IIA inhibited, or the phosphorylated form stimulated lactose counterflow, a LacS-IIA truncation mutant of LacS was constructed. Detailed analyses of transport in whole cells and in proteoliposomes indicated that unphosphorylated LacS-IIA does not functionally interact with the carrier domain. Instead, interaction of the phosphorylated form of LacS-IIA with the carrier stimulates lactose counterflow transport. The proposed mode of regulation thus proceeds via a mechanism opposite to the inducer exclusion type of regulation in Gram-negative bacteria, where transporters are inhibited by binding of the unphosphorylated form of IIAGlc.
Although the quaternary state has been assessed in detail for only a few members of the major facilitator superfamily (MFS), it is clear that multiple oligomeric states are represented within the ...MFS. One of its members, the lactose transporter LacS from
Streptococcus thermophilus assumes a dimeric structure in the membrane and
in vitro analysis showed functional interactions between both subunits when proton motive force (Δp)-driven transport was assayed. To study the interactions in further detail, a covalent dimer was constructed consisting of in tandem fused LacS subunits. These covalent dimers, composed of active and completely inactive subunits, were expressed in
Escherichia coli, and initial rates of Δp-driven lactose uptake and lactose counterflow were determined. We now show that also
in vivo, both subunits interact functionally; that is, partial complementation of the inactive subunit was observed for both transport modes. Thus, both subunits interact functionally in Δp-driven uptake and in counterflow transport. In addition, analysis of in tandem fused LacS subunits containing one regulatory LacS-IIA domain showed that regulation is primarily an intramolecular event.
A Missing Link in Membrane Protein Evolution Poolman, Bert; Geertsma, Eric R.; Slotboom, Dirk-Jan
Science (American Association for the Advancement of Science),
03/2007, Letnik:
315, Številka:
5816
Journal Article
Recenzirano
Odprti dostop
Discerning the orientation of subunits of an unusual bacterial membrane protein suggests how the particular topology of other membrane proteins may have evolved.
We present a generic method for the site‐specific and differential labeling of multiple cysteine residues in one protein. Phenyl arsenic oxide has been employed as a protecting group of two closely ...spaced thiols, allowing first labeling of a single thiol. Subsequently, the protecting group is removed, making available a reactive dithiol site for labeling with a second probe. For proof‐of‐principle, single and triple Cys mutants of the sulphate binding protein of an ABC transporter were constructed. The closely spaced thiols were engineered on the basis of the crystal structure of the protein and placed in different types of secondary structure elements and at different spacing. We show that phenyl arsenic oxide is a good protecting group for thiols spaced 6.3–7.3 Å. Proteins were labeled with two different fluorescent labels and the labeling ratios were determined with UV‐Vis spectroscopy and MALDI‐Tof mass spectrometry. The average labeling efficiency was ∼80% for the single thiol and 65–90% for the dithiol site.
The mannitol permease (EII
Mtl) from
Escherichia coli couples mannitol transport to phosphorylation of the substrate. Renewed topology prediction of the membrane-embedded C domain suggested that EII
...Mtl contains more membrane-embedded segments than the six proposed previously on the basis of a PhoA fusion study. Cysteine accessibility was used to confirm this notion. Since cysteine 384 in the cytoplasmic B domain is crucial for the phosphorylation activity of EII
Mtl, all cysteine mutants contained this activity-linked cysteine residue in addition to those introduced for probing the membrane topology of the protein. To distinguish between the activity-linked cysteine and the probed cysteine, either trypsin was used to specifically digest the two cytoplasmic domains (A and B), thereby removing Cys384, or Cys384 was protected by phosphorylation from alkylation by
N-ethylmaleimide (NEM). Our data show that upon phosphorylation EII
Mtl undergoes major conformational changes, whereby residues in the putative first cytoplasmic loop become accessible to NEM. Other residues in this loop were accessible to NEM in intact cells and inside-out membrane vesicles, but cysteine residues at these positions only reacted with the membrane-impermeable sulfhydryl reagent from the periplasmic side of the protein. These and other results suggest that the predicted loop between TM2 and TM3 may fold back into the membrane and form part of the translocation path.
As the equivalent to gatekeepers of the cell, membrane transport proteins perform a variety of critical functions. Progress on the functional and structural characterization of membrane proteins is ...slowed due to problems associated with their (heterologous) overexpression. Often, overexpression fails or leads to aggregated material from which the production of functionally refolded protein is challenging. It is still difficult to predict whether a given membrane protein can be overproduced in a functional competent state. As a result, the most straightforward strategy to set up an overexpression system is to screen a multitude of conditions, including the comparison of homologues, type and location of (affinity) tags, and distinct expression hosts. Here, we detail methodology to rapidly establish and optimize (membrane) protein expression in Escherichia coli and Lactococcus lactis.
The lactose transporter from
Streptococcus thermophilus catalyses the symport of galactosides and protons. The carrier domain of the protein harbours the contact sites for dimerization, and the ...individual subunits in the dimer interact functionally during the transport reaction. As a first step towards the elucidation of the mechanism behind the cooperation between the subunits, regions involved in the dimer interface were determined by oxidative and chemical cross-linking of 12 cysteine substitution mutants. Four positions in the protein were found to be susceptible to intermolecular cross-linking. To ensure that the observed cross-links were not the result of randomly colliding particles, the cross-linking was studied in samples in which either the concentration of LacS in the membrane was varied or the oligomeric state was manipulated. These experiments showed that the cross-links were formed specifically within the dimer. The four regions of the protein located at the dimer interface are close to the extracellular ends of transmembrane segments V and VIII and the intracellular ends of transmembrane segments VI and VII.
The SLC26 or SulP proteins constitute a large family of anion transporters that are ubiquitously expressed in pro- and eukaryotes. In human, SLC26 proteins perform important roles in ion homeostasis ...and malfunctioning of selected members is associated with diseases. This protocol details the production and crystallization of a prokaryotic SLC26 homolog, termed SLC26Dg, from Deinococcus geothermalis. Following these instructions we obtained well-folded and homogenous material of the membrane protein SLC26Dg and the nanobody Nb5776 that enabled us to crystallize the complex and determine its structure (Geertsma et al., 2015). The procedure may be adapted to purify and crystallize other membrane protein complexes.
Making your (Dsb) connection: the redox pathway bringing reducing equivalents from bacterial cytoplasm, across the inner membrane, to the three reductive Dsb pathways in the otherwise oxidizing ...periplasm (see scheme; TR=thioredoxin reductase, Trx=thioredoxin) is reconstituted from purified components. Transfer of reducing equivalents across the membrane is demonstrated and underlying mechanistic details are revealed.
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