Hydrophilic C-terminal Domain Research Articles

The short C-terminal hydrophilic domain of NhaH Na+/H+ antiporter from Halobacillus dabanensis with roles in resistance to salt and in pH sensing

The Na+/H+ antiporter plays key roles in maintaining low cytoplasmic Na+ level and pH homeostasis, while little is known about the Carboxyl-terminal hydrophilic tails of prokaryotic antiporters. In our previous study, the first Na+/H+ antiporter gene nhaH from moderate halophiles was cloned from Halobacillus dabanensis D-8 by functional complementation. A topological model suggested that only nine amino acid residues (395PLIKKLGMI403) existed in the hydrophilic C-terminal domain of NhaH. The C-terminal truncated mutant of NhaH was constructed by PCR strategy and designated as nhaHΔC. Salt tolerance experiment demonstrated that the deletion of hydrophilic C-terminal nine amino acid residues significantly inhibited the complementation ability of E. coli KNabc, in which three main Na+/H+ antiporters nhaA, nhaB and chaA were deleted. Everted membrane vesicles prepared from E. coli KNabc/nhaHΔC decreased both Na+/H+ and Li+/H+ exchange activities of NhaH, and also resulted in an acidic shift of its pH profile for Na+, indicating a critical role of the short C-terminal domain of NhaH antiporter in alkali cation binding/translocation and pH sensing.

Mrtl—A translation/localization regulatory protein encoded within the human c‐myc locus and distributed throughout the endoplasmic and nucleoplasmic reticular network

mrtl (myc-related translation/localization regulatory factor) is a previously uncharacterized protein synthesized from the first open reading frame contained within the human c-myc P0 transcript, approximately 800 nucleotides upstream of the Myc coding sequence. The mrtl protein, 114 amino acids in length, is projected to contain an N-terminal transmembrane domain and a highly charged C-terminal interaction domain with homology to numerous RNA-binding proteins. Using monoclonal antibodies raised against the hydrophilic C-terminal domain, endogenous mrtl was visualized in human breast tumor cell lines and primary mammary epithelial cells at the nuclear envelope and contiguous endoplasmic/nucleoplasmic reticulum. mrtl colocalizes and coimmunoprecipitates with translation initiation factor eIF2alpha and the 40S ribosomal protein RACK1, and appears capable of binding specifically to the c-myc RNA. Inducible ectopic overexpression of wild-type mrtl interferes with the function of endogenous mrtl, which results in loss of Myc from the nucleus. Furthermore, treatment of cells with a peptide derived from the C-terminal domain displaces endogenous mrtl and causes a dramatic reduction in total cellular Myc protein levels. Together with our previous work demonstrating complete loss of tumorigenicity in association with ectopic expression of the c-myc P0 5'-UTR (containing the mrtl coding sequence), these results suggest that mrtl may serve an important function in regulating Myc translation and localization to the nucleus, perhaps ultimately contributing to the role of the c-myc locus in oncogenesis.

The T4 RI Antiholin Has an N-Terminal Signal Anchor Release Domain That Targets It for Degradation by DegP

Lysis inhibition (LIN) of T4-infected cells was one of the foundational experimental systems for modern molecular genetics. In LIN, secondary infection of T4-infected cells results in a dramatically protracted infection cycle in which intracellular phage and endolysin accumulation can continue for hours. At the molecular level, this is due to the inhibition of the holin, T, by the antiholin, RI. RI is only 97 residues and contains an N-terminal hydrophobic domain and a C-terminal hydrophilic domain; expression of the latter domain fused to a secretory signal sequence is sufficient to impose LIN, due to its specific interaction with the periplasmic domain of the T holin. Here we show that the N-terminal sequence comprises a signal anchor release (SAR) domain, which causes the secretion of RI in a membrane-tethered form and then its subsequent release into the periplasm, without proteolytic processing. Moreover, the SAR domain confers both functional lability and DegP-mediated proteolytic instability on the released form of RI, although LIN is not affected in a degP host. These results are discussed in terms of a model for the activation of RI in the establishment of the LIN state.

Molecular cloning and expression analysis of cDNAs encoding a putative Nrt2 nitrate transporter from peach

Two cDNA clones encoding a putative high-affinity nitrate transporter (Nrt2) were isolated from a cDNA library of peach (Prunus persica (L.) Batsch) roots. The nucleotide sequences of the clones, named PpNrt2.1 and PpNrt2.2, showed 99% identity in their coding regions, but their 3'-untranslated sequences showed only 40% identity. The deduced amino acid sequences of the clones showed 44-90% homology to NRT2 proteins from other plants. Both clones contained open reading frames encoding polypeptides of 530 amino acid residues, each with a predicted molecular mass of about 57 kDa. Both polypeptides had 12 putative transmembrane domains and a long hydrophilic C-terminal domain like other members of this protein family. Analyses of the genomic structures and of Southern blots suggested that PpNrt2.1 and PpNrt2.2 originated from different loci in the peach genome. In Northern blots, transcripts of PpNrt2.1 and PpNrt2.2 were detected in roots but not shoots of seedlings after nitrate application. They were not detected in leaves, stems, flowers, fruits or seeds of mature trees in the field, indicating root-specific expression of PpNrt2.1 and PpNrt2.2. Application of nitrate to roots of nitrogen-deprived seedlings strongly induced expression of PpNrt2.1 within 0.5 h, and gradually induced expression of PpNrt2.2. Application of ammonium to roots of nitrogen-deprived seedlings repressed expression of PpNrt2.1 and PpNrt2.2. The relationship of PpNrt2.1 and PpNrt2.2 to the inducible high-affinity transport system (iHATS) involved in the uptake of nitrate in peach roots is discussed.

Genus specific features of bovine papillomavirus E6, E7, E5 and E8 proteins

Six bovine papillomavirus (BPV) types, BPV-1 to -6, have been classified in genera Delta-papillomavirus (BPV-1 and -2), Epsilon-papillomavirus (BPV-5) and Xi-papillomavirus (BPV-3, -4 and -6). In addition, 16 unclassified putative BPV types have been reported. In the present study, we characterized genus specific features of E6, E7, E5 (formerly E8) and E8 proteins of seven putative BPV types, BAPV-1, -2, -3, -4 and -10, BAA-5 and BPV-3c. These putative BPV types were classified in genera Epsilon- or Xi-papillomavirus. The E6 proteins of BPV and putative BPV types in Epsilon-papillomavirus showed high sequence similarities, and contained two typical zinc-binding domains. However, E7 proteins contained atypical zinc-binding domains, and lacked the canonical retinoblastoma tumor suppressor protein (pRB)-binding motif. BPV and putative BPV types in Xi-papillomavirus contained E5 or E8 open reading frame (ORF) in the E6 position. The E5 ORFs encoded proteins consist of 42-amino acid with a hydrophobic transmembrane and a hydrophilic C-terminal domain. But the E8 ORFs encoded protein which have two transmembrane domains. Our results demonstrated that E5, E8, E6, E7 proteins of the putative BPV types, which are presumably classified in genera Epsilon- or Xi-papillomavirus, retained the some genus specific features.

Functions of Y-box Binding Protein and Its Role in Tumori-genicity

Y-box binding proteins, belonging to a family of multifunctional proteins conserved from bacteria to human, are involved in transcriptional and translational regulation of various genes, mRNA alternative splicing, DNA replication and repair, as well as cell proliferation. A typical Y-box binding protein contains three structure domains, namely the N-terminal domain, the hydrophilic C-terminal domain and the conserved cold shock domain (CSD) which binds strongly to inverted CCAAT box found in different promoters and determines protein function.Y-box binding proteins may play an important physiological role in cell proliferation.For example, the human Y-box protein 1 (YB-1) may be repressed in the oncogenic phosphoinositide 3-kinase (PI3K) pathway. In addition, it may also act as a negative regulator of p53. It has been demonstrated that YB-1 represses transcription of the p53 promoter in a sequence-specific manner using specifically reporter assays. This implies that YB-1 may, in some situations, protect cells from p53-mediated apoptosis, indicating that YB-1 may be a good target for the development of a new therapeutics.The function of Y-box binding protein and its effect on carcinogenesis are summarized in the paper. We hope to further explore the functional roles of Y-box binding protein, and provide some helpful lines and suggestions for tumor control.

FlhB Regulates Ordered Export of Flagellar Components via Autocleavage Mechanism

The bacterial flagellum is a predominantly cell-external super-macromolecular construction whose structural components are exported by a flagellum-specific export apparatus. One of the export apparatus proteins, FlhB, regulates the substrate specificity of the entire apparatus; i.e. it has a role in the ordered export of the two main groups of flagellar structural proteins such that the cell-proximal components (rod-/hook-type proteins) are exported before the cell-distal components (filament-type proteins). The controlled switch between these two export states is believed to be mediated by conformational changes in the structure of the C-terminal cytoplasmic domain of FlhB (FlhB(C)), which is consistently and specifically cleaved into two subdomains (FlhB(CN) and FlhB(CC)) that remain tightly associated with each other. The cleavage event has been shown to be physiologically significant for the switch. In this study, the mechanism of FlhB cleavage has been more directly analyzed. We demonstrate that cleavage occurs in a heterologous host, Saccharomyces cerevisiae, deficient in vacuolar proteinases A and B. In addition, we find that cleavage of a slow-cleaving variant, FlhB(C)(P270A), is stimulated in vitro at alkaline pH. We also show by analytical gel-filtration chromatography and analytical ultracentrifugation experiments that both FlhB(C) and FlhB(C)(P270A) are monomeric in solution, and therefore self-proteolysis is unlikely. Finally, we provide evidence via peptide analysis and FlhB cleavage variants that the tertiary structure of FlhB plays a significant role in cleavage. Based on these results, we propose that FlhB cleavage is an autocatalytic process.

Membrane Topology of the STT3 Subunit of the Oligosaccharyl Transferase Complex

The highly conserved membrane protein STT3 is part of the oligosaccharyl transferase complex in the endoplasmic reticulum of eukaryotic cells. Various experimental observations strongly suggest that STT3 contains the active site of the complex. Here, we report a detailed topology study of STT3 from two different organisms, Saccharomyces cerevisiae and mouse, using in vivo and in vitro topology mapping assays. Our results suggest that STT3 has 11 transmembrane helices and an overall N(cyt)-C(lum) orientation.

Identification of a domain in the α‐subunit of the oxaloacetate decarboxylase Na+ pump that accomplishes complex formation with the γ‐subunit

The oxaloacetate decarboxylase Na+ pumps OAD-1 and OAD-2 of Vibrio cholerae are composed of a peripheral alpha-subunit associated with two integral membrane-bound subunits (beta and gamma). The alpha-subunit contains the carboxyltransferase domain in its N-terminal portion and the biotin-binding domain in its C-terminal portion. The gamma-subunit plays a profound role in the assembly of the complex. It interacts with the beta-subunit through its N-terminal membrane-spanning region and with the alpha-subunit through its hydrophilic C-terminal domain. The biochemical and structural requirements for the latter interaction were analysed with OAD-2 expression clones for subunit alpha-2 and the C-terminal domain of gamma-2, termed gamma'-2. If the two proteins were synthesized together in Escherichia coli they formed a complex that was stable at neutral pH and dissociated at pH<5.0. An internal stretch of 40 amino acids of alpha-2 was identified by deletion mutagenesis to be essential for the binding with gamma'-2. This portion of the alpha-subunit is connected via flexible linker peptides to the carboxyltransferase domain at its N terminus and to the biotin-binding domain at its C terminus. Results of site-directed mutagenesis indicated that a conserved tyrosine (491) and threonine 494 of this peptide contributed significantly to the stability of the complex with gamma'-2. This peptide therefore represents a newly identified, separate domain of the alpha-subunit and has been called the 'association domain'.

CcmD Is Involved in Complex Formation between CcmC and the Heme Chaperone CcmE during Cytochrome c Maturation

CcmD is a small membrane protein involved in heme delivery to the heme chaperone CcmE during cytochrome c maturation. Here we show that it physically interacts with CcmE and CcmC, another essential component of the heme delivery system. We demonstrate the formation of a ternary complex consisting of CcmCDE. A deletion analysis of individual domains revealed that the central hydrophobic domain is essential for its function. Moreover, the C-terminal, cytoplasmic domain seems to require a net positive charge to be functional. Our topology analysis indicates that CcmD is an integral interfacial membrane protein with its N and C termini extruding into the cytoplasmic side of the membrane. Interactions of CcmD with either ferrochelatase, the last heme biosynthetic enzyme, or directly with heme were not detectable. We postulate a function for CcmD in protein-protein interaction or membrane protein assembly required for the heme delivery process.

The effect of recombinant mouse amelogenins on the formation and organization of hydroxyapatite crystals in vitro

Amelogenin is the most abundant protein in developing dental enamel. It is believed to play an important role in the regulation of the growth and organization of enamel crystals. Amelogenin, unlike many other proteins found in biominerals, is mostly hydrophobic except for a 13 amino acid hydrophilic C-terminal domain. To clarify the role of amelogenin in enamel mineralization, we designed calcium phosphate crystal growth experiments in the presence of recombinant amelogenins with or without the charged C-terminal domain. The shape and organization of the crystals were examined by TEM in bright field and diffraction modes. It was found that both full-length and truncated amelogenin inhibit crystal growth in directions normal to the c-axis. At the same time, crystallites organized into parallel arrays only in the presence of the full-length amelogenin in monomeric form. Pre-assembled amelogenins had no effect on crystals organization. These results imply that the hydrophobic portion of amelogenin plays a role in an inhibition of crystal growth, whereas the C-terminal domain is essential for the alignment of crystals into parallel arrays. Our data also suggest that nascent enamel structure emerges as a result of cooperative interactions between forming crystals and assembling proteins.

Calmodulin-binding proteins in bryophytes: identification of abscisic acid-, cold-, and osmotic stress-induced genes encoding novel membrane-bound transporter-like proteins

Plant responses to environmental stresses are mediated in part by signaling processes involving cytosolic Ca 2+ and a Ca 2+-binding protein, calmodulin. Screening with radiolabeled calmodulin of a cDNA library of the moss Physcomitrella patens resulted in identification of genes encoding novel membrane transporter-like proteins, MCamb1 and MCamb2. These proteins each had a central hydrophobic domain with two putative membrane spans and N- and C-terminal hydrophilic domains, and showed sequence similarity to mammalian inward rectifier potassium channels. Calmodulin binds to MCamb1 and MCamb2 via interaction with basic amphiphilic amino acids in the C-terminal domain. Levels of MCamb1 and MCamb2 transcripts increased dramatically following treatment with low temperature, hyperosmotic solutes, and the stress hormone abscisic acid, all of which were previously shown to increase cellular tolerance to freezing stress. These results suggest that calmodulin participates in cellular signaling events leading to enhancement of stress resistance through regulation of novel transporter-like proteins.

Topological analysis of a plant vacuolar Na+/H+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity.

We conducted an analysis of the topology of AtNHX1, an Arabidopsis thaliana vacuolar Na+/H+ antiporter. Several hydrophilic regions of the antiporter were tagged with a hemagglutinin epitope, and protease protection assays were conducted to determine the membrane topology of the antiporter by using yeast as a heterologous expression system. The overall structure of AtNHX1 is distinct from the human Na+/H+ antiporter NHE1 or any known Na+/H+ antiporter. It is comprised of nine transmembrane domains and a hydrophilic C-terminal domain. Three hydrophobic regions do not appear to span the tonoplast membrane, yet appear to be membrane associated. Our results also indicate that, whereas the N terminus of AtNHX1 is facing the cytosol, almost the entire C-terminal hydrophilic region resides in the vacuolar lumen. Deletion of the hydrophilic C terminus resulted in a dramatic increase in the relative rate of Na+/H+ transport. The ratio of Na+/K+ transport was twice that of the unmodified AtNHX1. This altered ratio resulted from a relatively small decrease in K+/H+ transport with a large increase in Na+/H+ transport. The vacuolar localization of the C terminus of the AtNHX1, taken together with the regulation of the antiporter selectivity by its C terminus, demonstrates the existence of luminal vacuolar regulatory mechanisms of the antiporter activity.

Acute regulation of the SLC26A3 congenital chloride diarrhoea anion exchanger (DRA) expressed in Xenopus oocytes.

Mutations in the human SLC26A3 gene, also known as down-regulated in adenoma (hDRA), cause autosomal recessive congenital chloride-losing diarrhoea (CLD). hDRA expressed in Xenopus oocytes mediated bidirectional Cl--Cl- and Cl--HCO3- exchange. In contrast, transport of oxalate was low, and transport of sulfate and of butyrate was undetectable. Two CLD missense disease mutants of hDRA were nonfunctional in oocytes. Truncation of up to 44 C-terminal amino acids from the putatively cytoplasmic C-terminal hydrophilic domain left transport function unimpaired, but deletion of the adjacent STAS (sulfate transporter anti-sigma factor antagonist) domain abolished function. hDRA-mediated Cl- transport was insensitive to changing extracellular pH, but was inhibited by intracellular acidification and activated by NH4+ at acidifying concentrations. These regulatory responses did not require the presence of either hDRA's N-terminal cytoplasmic tail or its 44 C-terminal amino acids, but they did require more proximate residues of the C-terminal cytoplasmic domain. Although only weakly sensitive to inhibition by stilbenes, hDRA was inhibited with two orders of magnitude greater potency by the anti-inflammatory drugs niflumate and tenidap. cAMP-insensitive Cl--HCO3- exchange mediated by hDRA gained modest cAMP sensitivity when co-expressed with cystic fibrosis transmembrane conductance regulator (CFTR). Despite the absence of hDRA transcripts in human cell lines derived from CFTR patients, DRA mRNA was present at wild-type levels in proximal colon and nearly so in the distal ileum of CFTR(-/-) mice. Thus, pharmacological modulation of DRA might be a useful adjunct treatment of cystic fibrosis.

Characterization of homologues of the apple proliferation immunodominant membrane protein gene from three related phytoplasmas

SUMMARY Homologues of the immunodominant membrane protein gene from apple proliferation (AP) phytoplasma have been cloned and sequenced for three further members of the AP subclade, namely European stone fruit yellows, peach yellow leaf roll and a European isolate of pear decline (PD). The putative translation products of all three were similar in size to that of AP and all had a transmembrane region towards the N-terminus and a large C-terminal hydrophilic domain, probably held on the outside of the cell membrane in vivo. Sequence similarities for the putative proteins were compared with interrelationships of the phytoplasmas as measured by rRNA gene sequence similarity. The proteins from AP and PD were more similar (57% identical in the major hydrophilic domain) than those for any other pair (31-34%), but these two phytoplasmas were not more closely related by rRNA gene sequences than other pairs. The possibility that the relative similarities of these proteins is related to the host is discussed. It is suggested that the similarity of the AP and PD proteins may reflect the fact that these two proteins have narrow plant host ranges in two closely related genera in the tribe Maloideae (family Rosaceae), whilst the other two have broader host ranges, mainly in the tribe Prunoideae.

Structure and regulation of expression of the mouse GH receptor.

GH-binding protein (GHBP) in the mouse consists of a ligand-binding domain, which is identical to the extracellular portion of the GH receptor (GHR), and a hydrophilic C-terminal domain, in place of the transmembrane and intracellular domains of the GHR. The two proteins are encoded by separate mRNAs which are derived from a single gene by alternative splicing. Determination of the gestational profiles of GHR and GHBP mRNA expression in mouse liver and placenta shows that in the liver, the 1.4 kb mRNA corresponding to the mouse GHBP increases approximately 20-fold between non-pregnant and late pregnant mice, whereas the relative increase in the expression of the 4.2 kb mouse GHR was 8-fold. The rise in the steady-state levels of both mRNAs began on day 9 of gestation. Mouse GHBP mRNA levels continue to rise until day 15 of pregnancy, while GHR mRNA abundance reaches a plateau by day 13. By elucidating the temporal changes in GHR and GHBP mRNA abundance during pregnancy and lactation in multiple maternal tissues and by assessing the ontogeny of these mRNAs in fetal and early postnatal mouse liver, our studies have demonstrated that the alternative splicing of mouse GHR/GHBP mRNA precursor is regulated in a tissue-, developmental stage- and physiological state-specific manner. In vitro studies using hepatocytes in culture have begun to elucidate the hormonal factor(s) involved in the gestation control of the expression of GHR and GHBP. Treatment of hepatocytes with GH or estradiol (E2) alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR mRNA concentrations were also up-regulated 2- to 3-fold. ICI 182-780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2- and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes treated with E2 and GH compared with those in untreated cells or cells treated with either E2 or GH alone. Our studies in the mouse suggest that GHBP is an important cell-surface receptor for GH in the liver. These studies postulate that an arginine-glycine-aspartic acid sequence found on mouse GHBP but absent in other species is responsible for the association of GHBP with the plasma membrane by binding to one or more integrins on the surface of liver cells.

Targeting and membrane-insertion of a sunflower oleosin in vitro and in Saccharomyces cerevisiae: the central hydrophobic domain contains more than one signal sequence, and directs oleosin insertion into the endoplasmic reticulum membrane using a signal anchor sequence mechanism.

A range of N- and C-terminal deletions of an oleosin from Helianthus annuus L. were used to study the endoplasmic reticulum (ER) targeting and membrane insertion of this protein both in vitro and in vivo in yeast ( Saccharomyces cerevisiae). Neither the N- nor the C-terminal hydrophilic domains are important for targeting and/or membrane insertion, with all the information required for these processes located within the central hydrophobic region of the protein. However, in vitro membrane-insertion experiments suggest that these domains are important for a correct topology of the oleosin within the ER membrane. The first half of the hydrophobic central domain, flanked by the positively charged N-terminal domain, is likely to function as a type-II signal-anchor (SAII) sequence. However, in the absence of the N-terminal 26 residues of this domain, the proline-knot region and the second half of this hydrophobic domain are sufficient to direct oleosin to the ER and to allow stable (but far less efficient) integration of the protein into the membrane. Taken together, these results indicate that oleosin contains more than one domain that is capable of interacting with the signal recognition particle to direct the protein to the ER membrane.

Structural Studies of the HIV-1 Accessory Protein Vpu in Langmuir Monolayers: Synchrotron X-ray Reflectivity

Vpu is an 81 amino acid integral membrane protein encoded by the HIV-1 genome with a N-terminal hydrophobic domain and a C-terminal hydrophilic domain. It enhances the release of virus from the infected cell and triggers degradation of the virus receptor CD4. Langmuir monolayers of mixtures of Vpu and the phospholipid 1,2-dilignoceroyl- sn-glycero-3-phosphocholine (DLgPC) at the water–air interface were studied by synchrotron radiation-based x-ray reflectivity over a range of mole ratios at constant surface pressure and for several surface pressures at a maximal mole ratio of Vpu/DLgPC. Analysis of the x-ray reflectivity data by both slab model-refinement and model-independent box-refinement methods firmly establish the monolayer electron density profiles. The electron density profiles as a function of increasing Vpu/DLgPC mole ratio at a constant, relatively high surface pressure indicated that the amphipathic helices of the cytoplasmic domain lie on the surface of the phospholipid headgroups and the hydrophobic transmembrane helix is oriented approximately normal to the plane of monolayer within the phospholipid hydrocarbon chain layer. At maximal Vpu/DLgPC mole ratio, the tilt of the transmembrane helix with respect to the monolayer normal decreases with increasing surface pressure and the conformation of the cytoplasmic domain varies substantially with surface pressure.

Influence of alpha-helices on the emulsifying properties of proteins.

A peptide derived from apomyoglobin by cyanogen bromide cleavage was found to be an active emulsifier. This molecule, peptide 1-55, has two potential amphipathic alpha-helices and a hydrophilic C-terminal domain. The importance of each of these domains to the emulsifying properties of this molecule was investigated by testing the products of gene constructs based on the sequence of peptide 1-55, but lacking one of the three domains. The emulsifying activity of the peptides lacking either of the alpha-helices was correlated with the hydrophobic moments of their respective helices. The hydrophobic moment is a measure of the amphipathicity of alpha-helices; a hydrophobic moment analysis of other emulsifying peptides supports the hypothesis that a high hydrophobic moment contributes to good emulsifying properties in a molecule which contains alpha-helices.

HPr(His∼P)-mediated Phosphorylation Differently Affects Counterflow and Proton Motive Force-driven Uptake via the Lactose Transport Protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus has a C-terminal hydrophilic domain that is homologous to IIA protein and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The IIA domain of LacS is phosphorylated on His-552 by the general energy coupling proteins of the PTS, which are Enzyme I and HPr. To study the effect of phosphorylation on transport, the LacS protein was purified and incorporated into liposomes with the IIA domain facing outwards. This allowed the phosphorylation of the membrane-reconstituted protein by purified HPr(His approximately P) of S. thermophilus. Phosphorylation of LacS increased the V(max) of counterflow transport, whereas the V(max) of the proton motive force (delta p)-driven lactose uptake was not affected. In line with a range of kinetic studies, we propose that phosphorylation affects the rate constants for the reorientation of the ternary complex (LacS with bound lactose plus proton), which is rate-determining for counterflow but not for delta p-driven transport.