|
|
|
|||||||
|
|||||||||
(Received for publication, February 9, 1996, and in revised form, April 4, 1996)
From the The lactose transport protein (LacS) of
Streptococcus thermophilus was amplified to levels as high
as 8 and 30% of total membrane protein in Escherichia coli
and S. thermophilus, respectively. In both organisms the
protein was functional and the expression levels were highest with the
streptococcal lacS promoter. Also a LacS deletion mutant,
lacking the carboxyl-terminal regulatory domain, could be amplified to
levels >20% of membrane protein. Membranes from S. thermophilus proved to be superior in terms of efficient
solubilization and ease and extent of purification of LacS; >95% of
LacS was solubilized with relatively low concentrations of Triton
X-100, n-octyl-
Volume 271, Number 26,
Issue of June 28, 1996
pp. 15358-15366
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,
,
and
Department of Microbiology, Groningen
Biomolecular Sciences and Biotechnology Institute, University of
Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands, the
§ Department of Biochemistry and Molecular Biology,
University of Leeds, Leeds LS2 9JT, United Kingdom, and the
Laboratoire J. Maetz, Département de Biologie Cellulaire
et Moléculaire du Commissariat à l'Energie Atomique,
06230 Villefranche sur mer, France
-D-glucoside,
n-dodecyl--D-maltoside, or
C12E8. The LacS protein carrying a
poly-histidine tag was purified in large quantities (~5 mg/liter of
culture) and with a purity >98% in a two-step process involving
nickel chelate affinity and anion exchange chromatography. The membrane
reconstitution of LacS was studied systematically by stepwise
solubilization of preformed liposomes, prepared from E. coli phospholipid and phosphatidylcholine, and protein
incorporation at the different stages of liposome solubilization. The
detergents were removed by adsorption onto polystyrene beads and
H+-lactose symport and lactose counterflow were measured.
Highest transport activities were obtained when Triton X-100 was used
throughout the solubilization/purification procedure, whereas activity
was lost irreversibly with
n-octyl--D-glucoside. For reconstitutions
mediated by n-dodecyl--D-maltoside,
C12E8, and to a lesser extent Triton X-100, the
highest transport activities were obtained when the liposomes were
titrated with low amounts of detergent (onset of liposome
solubilization). Importantly, under these conditions proteoliposomes
were obtained in which LacS was reconstituted in an inside-out
orientation, as suggested by the outside labeling of a single cysteine
mutant with a membrane impermeable biotin-maleimide. The results are
consistent with a mechanism of reconstitution in which the hydrophilic
regions of LacS prevent a random insertion of the protein into the
membrane. Consistent with the in vivo lactose/galactose
exchange catalyzed by the LacS protein, the maximal rate of lactose
counterflow was almost 2 orders of magnitude higher than that of
H+-lactose symport.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  N. A. B. N. Mahmood, E. Biemans-Oldehinkel, J. S. Patzlaff, G. K. Schuurman-Wolters, and B. Poolman Ion Specificity and Ionic Strength Dependence of the Osmoregulatory ABC Transporter OpuA J. Biol. Chem., October 6, 2006; 281(40): 29830 - 29839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Meyer-Lipp, N. Sery, C. Ganea, C. Basquin, K. Fendler, and G. Leblanc The Inner Interhelix Loop 4-5 of the Melibiose Permease from Escherichia coli Takes Part in Conformational Changes after Sugar Binding J. Biol. Chem., September 8, 2006; 281(36): 25882 - 25892. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Hall and A. M. Pajor Functional Characterization of a Na+-Coupled Dicarboxylate Carrier Protein from Staphylococcus aureus J. Bacteriol., August 1, 2005; 187(15): 5189 - 5194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-J. Liang, K. J. Wilson, H. Xie, J. Knol, S. Suzuki, N. G. Rutherford, P. J. F. Henderson, and R. A. Jefferson The gusBC Genes of Escherichia coli Encode a Glucuronide Transport System J. Bacteriol., April 1, 2005; 187(7): 2377 - 2385. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Segawa, R. P. Soares, M. Kawakita, S. M. Beverley, and S. J. Turco Reconstitution of GDP-mannose Transport Activity with Purified Leishmania LPG2 Protein in Liposomes J. Biol. Chem., January 21, 2005; 280(3): 2028 - 2035. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Gendreau, S. Voswinkel, D. Torres-Salazar, N. Lang, H. Heidtmann, S. Detro-Dassen, G. Schmalzing, P. Hidalgo, and C. Fahlke A Trimeric Quaternary Structure Is Conserved in Bacterial and Human Glutamate Transporters J. Biol. Chem., September 17, 2004; 279(38): 39505 - 39512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. K. Doeven, R. Abele, R. Tampe, and B. Poolman The Binding Specificity of OppA Determines the Selectivity of the Oligopeptide ATP-binding Cassette Transporter J. Biol. Chem., July 30, 2004; 279(31): 32301 - 32307. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Searls, X. Chen, S. Allen, and M. D. Yudkin Evaluation of the Kinetic Properties of the Sporulation Protein SpoIIE of Bacillus subtilis by Inclusion in a Model Membrane J. Bacteriol., May 15, 2004; 186(10): 3195 - 3201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Lessard, A. Cochu, J.-D. Lemay, D. Roy, K. Vaillancourt, M. Frenette, S. Moineau, and C. Vadeboncoeur Phosphorylation of Streptococcus salivarius Lactose Permease (LacS) by HPr(His~P) and HPr(Ser-P)(His~P) and Effects on Growth J. Bacteriol., December 1, 2003; 185(23): 6764 - 6772. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Putman, H. W. van Veen, and W. N. Konings Molecular Properties of Bacterial Multidrug Transporters Microbiol. Mol. Biol. Rev., December 1, 2000; 64(4): 672 - 693. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. T. C. van den Bogaard, M. Kleerebezem, O. P. Kuipers, and W. M. de Vos Control of Lactose Transport, beta -Galactosidase Activity, and Glycolysis by CcpA in Streptococcus thermophilus: Evidence for Carbon Catabolite Repression by a Non-Phosphoenolpyruvate-Dependent Phosphotransferase System Sugar J. Bacteriol., November 1, 2000; 182(21): 5982 - 5989. [Abstract] [Full Text]
|
|
|
|
 |
|
 T. van der Heide and B. Poolman Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane PNAS, June 20, 2000; 97(13): 7102 - 7106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Fang, W. N. Konings, and B. Poolman Kinetics and Substrate Specificity of Membrane-Reconstituted Peptide Transporter DtpT of Lactococcus lactis J. Bacteriol., May 1, 2000; 182(9): 2530 - 2535. [Abstract] [Full Text]
|
|
|
|
|
|
A. Picon, E. R. S. Kunji, F. C. Lanfermeijer, W. N. Konings, and B. Poolman Specificity Mutants of the Binding Protein of the Oligopeptide Transport System of Lactococcus lactis J. Bacteriol., March 15, 2000; 182(6): 1600 - 1608. [Abstract] [Full Text]
|
|
|
|
|
|
L. M. Veenhoff and B. Poolman Substrate Recognition at the Cytoplasmic and Extracellular Binding Site of the Lactose Transport Protein of Streptococcus thermophilus J. Biol. Chem., November 19, 1999; 274(47): 33244 - 33250. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. W. Gunnewijk, P. W. Postma, and B. Poolman Phosphorylation and Functional Properties of the IIA Domain of the Lactose Transport Protein of Streptococcus thermophilus J. Bacteriol., January 15, 1999; 181(2): 632 - 641. [Abstract] [Full Text]
|
|
|
|
|
|
M.-C. Fann and P. C. Maloney Functional Symmetry of UhpT, the Sugar Phosphate Transporter of Escherichia coli J. Biol. Chem., December 11, 1998; 273(50): 33735 - 33740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Veenhoff, E. R. Geertsma, J. Knol, and B. Poolman Close Approximation of Putative alpha -Helices II, IV, VII, X, and XI in the Translocation Pathway of the Lactose Transport Protein of Streptococcus thermophilus J. Biol. Chem., July 28, 2000; 275(31): 23834 - 23840. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. W. Gunnewijk and B. Poolman Phosphorylation State of HPr Determines the Level of Expression and the Extent of Phosphorylation of the Lactose Transport Protein of Streptococcus thermophilus J. Biol. Chem., October 27, 2000; 275(44): 34073 - 34079. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. W. Gunnewijk and B. Poolman HPr(His~P)-mediated Phosphorylation Differently Affects Counterflow and Proton Motive Force-driven Uptake via the Lactose Transport Protein of Streptococcus thermophilus J. Biol. Chem., October 27, 2000; 275(44): 34080 - 34085. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. H. E. Friesen, J. Knol, and B. Poolman Quaternary Structure of the Lactose Transport Protein of Streptococcus thermophilus in the Detergent-solubilized and Membrane-reconstituted State J. Biol. Chem., October 20, 2000; 275(43): 33527 - 33535. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. H. M. L. Heuberger, E. Smits, and B. Poolman Xyloside Transport by XylP, a Member of the Galactoside-Pentoside-Hexuronide Family J. Biol. Chem., September 7, 2001; 276(37): 34465 - 34472. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |