Mechanism of lactose transport in Escherichia coli membrane vesicles: evidence for the involvement of a histidine residue(s) in the response of the lac carrier to the proton electrochemical gradient

García, María L.; Patel, Lekha; Padan, Etana; Kaback, H. Ronald

doi:10.1021/bi00266a012

Cited by 42 publications

(30 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The results presented here provide strong confirmation of previous studies (23)(24)(25) demonstrating that acylation of right-side-out membrane vesicles with diethylpyrocarbonate or photooxidation in the presence of rose bengal inactivates lactose/H' symport via the lac permease. Since these operations are known to be relatively specific for histidine residues and subsequent studies (25) with purified, reconstituted lac permease show that rose bengal-catalyzed photooxidation modifies two of the four histidine residues in the permease, it was concluded that histidine residues are critical for symport.…”

Section: Discussionsupporting

confidence: 90%

“…In other words, based on these preliminary results, it appears that permease containing arginine at position 322 is able to facilitate lactose movements down a concentration gradient at high substrate concentrations, while permease containing arginine at position 205 is completely inactive. Interestingly, treatment of right-side-out vesicles with diethylpyrocarbonate also inactivates lactose/H+ symport with little effect on downhill movement of lactose at high substrate concentrations (i.e., facilitated diffusion) (23)(24)(25). In this regard, direct measurements of facilitated diffusion in proteoliposomes reconstituted with the altered permease molecules should be informative (10).…”

Section: Discussionmentioning

confidence: 99%

“…Although the mechanism by which the lac permease transduces A!iH+ into a lactose concentration gradient is unknown, studies (23)(24)(25) with diethylpyrocarbonate and rose bengal in right-side-out membrane vesicles suggest that histidine residues may play an important role in coupling fl+ and lactose translocation. Site-directed mutagenesis (26) provides a means of altering amino acid residues in a protein with a high degree of specificity, and the approach has been used recently (27,28) to evaluate the role of cysteine-148 in the permease.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.

Padan¹,

Sarkar²,

Viitanen³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The lacY gene of Escherichia coil, which encodes the lac permease, has been modified by oligonucleotide-directed, site-specific mutagenesis such that each of the four histidine residues in the molecule is replaced with an arginine residue. Replacement of histidine-35 and histidine-39 with arginine has no apparent effect on permease activity. In contrast, replacement of either histidine-205 or histidine-322 by arginine causes a dramatic loss of transport activity, although the cells contain a normal complement of permease molecules, as determined by immunoadsorption assays. Interestingly, although substitution of histidine-205 or histidine-322 by arginine results in the loss of ability to catalyze active lactose transport, permease molecules with arginine at residue 322 appear to facilitate downhill lactose movements at high concentrations of the disaccharide. The results provide strong support for the contention that histidine residues in the lac permease play an important role in the coupling between lactose and proton translocation.The lac permease ofEscherichia coli is an intrinsic membrane protein encoded by the lacY gene that mediates symport (co-transport) of ,-galactosides with H' (cf. refs. 1-3 for recent reviews). Thus, in the presence of a H' electrochemical gradient (AAH+-interior negative and/or alkaline), the permease catalyzes accumulation of lactose against a concentration gradient. Conversely, under nonenergized conditions, downhill movement of /3-galactosides along a concentration gradient drives uphill translocation of H' with generation of A/.H+-The lacY gene has been cloned (4) and sequenced (5), and the permease has been purified to homogeneity in a completely functional state (6-14). Since proteoliposomes containing purified permease catalyze all of the reactions typical of (-galactoside with a high degree of specificity, and the approach has been used recently (27,28) to evaluate the role of cysteine-148 in the permease. We have now utilized the technique to systematically replace the four histidine residues in the permease with arginine, and the results are described herein.

show abstract

Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.

Padan¹,

Sarkar²,

Viitanen³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…x e preceding paper (Puttner et al, 1989) confirms and extends earlier observations (Padan et al, 1979(Padan et al, , 1985Patel et al, 1982;Garcia et al, 1982;Piittner et al, 1986) focusing on the importance of His322 in lactose/H+ symport by the lac permease. Thus, evidence was presented indicating that His322 may be directly involved in lactose-coupled H+ translocation.…”

supporting

confidence: 85%

Characterization of site-directed mutants in the lac permease of Escherichia coli. 2. Glutamate-325 replacements

Carrasco¹,

Püttner²,

Antes³

et al. 1989

Biochemistry

Self Cite

105

147

View full text Add to dashboard Cite

“…In particular, His322, which is located one helix-turn toward the periplasm from Glu325, is another functionally irreplaceable residue (49)(50)(51)(52)(53). In contrast to Glu325 mutants, exchange and counterflow are blocked in His322 mutants, and replacement with Asn or Gln results in a major decrease in affinity for galactosides (52,54).…”

Section: An Overall Mechanism For Coupling In Lacymentioning

confidence: 99%

Evolutionary mix-and-match with MFS transporters II

Madej

Kaback

2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

One fundamentally important problem for understanding the mechanism of coupling between substrate and H + translocation with secondary active transport proteins is the identification and physical localization of residues involved in substrate and H + binding. This information is exceptionally difficult to obtain with the Major Facilitator Superfamily (MFS) because of the broad sequence diversity of the members. The MFS is the largest and most diverse group of transporters, many of which are clinically important, and includes members from all kingdoms of life. A wide range of substrates is transported, in many instances against a concentration gradient by transduction of the energy stored in an H + electrochemical gradient using symport mechanisms, which are discussed herein. Crystallographic structures of MFS members indicate that a deep central hydrophilic cavity surrounded by 12 mostly irregular transmembrane helices represents a common structural feature. An inverted triple-helix structural symmetry motif within the N-and C-terminal six-helix bundles suggests that the proteins may have arisen by intragenic multiplication. In the work presented here, the triple-helix motifs are aligned in combinatorial fashion so as to detect functionally homologous positions with known atomic structures of MFS members. Substrate and H + -binding sites in symporters that transport substrates, ranging from simple ions like phosphate to more complex peptides or disaccharides, are found to be in similar locations. It also appears likely that there is a homologous ordered kinetic mechanism for the H + -coupled MFS symporters.membrane transport | sequence alignment | bioenergetics

show abstract

Mechanism of lactose transport in Escherichia coli membrane vesicles: evidence for the involvement of a histidine residue(s) in the response of the lac carrier to the proton electrochemical gradient

Cited by 42 publications

References 41 publications

Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.

Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.

Characterization of site-directed mutants in the lac permease of Escherichia coli. 2. Glutamate-325 replacements

Evolutionary mix-and-match with MFS transporters II

Contact Info

Product

Resources

About