![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Journal of Bacteriology, February 1999, p. 1039-1042, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Topology of RbsC, a Membrane Component of the
Ribose Transporter, Belonging to the AraH Superfamily
Yongkyu
Park and
Chankyu
Park*
National Creative Research Initiative Center
for Behavioral Genetics, Department of Biological Sciences, Korea
Advanced Institute of Science and Technology, Yusong-Ku, Taejon,
Republic of Korea
Received 14 September 1998/Accepted 9 November 1998
 |
ABSTRACT |
RbsC of Escherichia coli is the hydrophobic membrane
component of ribose uptake system classified as the ATP-binding
cassette transporter. To understand the structure and function of RbsC, its transmembrane topology was investigated by using 64 RbsC-PhoA fusions isolated either specifically or randomly. In order to confirm
the cytoplasmic location of the short C-terminal region (5 amino
acids), inside-out or right-side-out membrane vesicles were generated,
and the C-terminal region was found to be digested by carboxypeptidase
A only in inside-out vesicles. This result is consistent with the
model, based on the results of alkaline phosphatase fusions, in which
the protein traverses the membrane six times and the N and C termini
are exposed to the cytoplasm.
| |
TEXT |
The bacterial periplasmic binding
protein-dependent ATP-binding cassette (ABC) transporters are versatile
in substrate specificity, e.g., sugars, amino acids, oligopeptides, and
ions, etc., and generally consist of three to five proteins that fall
into three different functional categories (10). The first
component is the periplasmic substrate binding protein, which has two
separate domains with a substrate binding cleft in the middle
(16). The second component, consisting of either one or two
homologous proteins, is a hydrophobic membrane protein that possesses a
short conserved hydrophilic segment, containing a consensus EAA-G
motif, located at approximately 100 amino acids (aa) from the C
terminus (22). The hydrophilic third component has a highly
conserved ATP-binding motif (Walker A and B boxes) and the conserved
helical domain for interaction with the EAA-G region of the membrane
component (15).
Topologies of the inner membrane components of the binding protein
transporters have been determined by gene fusion and/or proteolysis
techniques. The OppB and OppC proteins involved in oligopeptide
transport (17) and MalG for maltose transport (5) were found to have six transmembrane (TM) helices with both ends oriented toward the cytoplasm. The MalF protein (514 aa), interacting with MalG for transport, comprises eight membrane-spanning segments with both ends of the protein exposed to the cytoplasm (3). ProW involved in the uptake of glycine betaine/proline has been reported to contain seven membrane-spanning segments with the C
terminus inside the cytoplasm and the large N-terminal extension (100 aa) located within the periplasmic space (7). HisQ (228 aa)
and HisM (235 aa) have five TM helices with periplasmic and cytoplasmic
N and C termini, respectively (13). However, of a group of
permeases known as the AraH/RbsC superfamily (21), transporting sugars such as arabinose, ribose, methylgalactoside, xylose, and allose, none were characterized for their membrane structures.
Ribose is transported in Escherichia coli through the
high-affinity, binding-protein-dependent system encoded by genes in the
rbs region of the chromosome (1). The system is
composed of the periplasmic ribose-binding protein (RbsB), the
hydrophobic membrane protein (RbsC), and the hydrophilic ATP-binding
protein (RbsA). Other AraH transporters (currently more than 20 are in the database) have similar organizations with periplasmic, membrane, and ATPase components (12, 14, 19, 23). Information on the
structure of the membrane component is a key to an understanding of the
mechanism of sugar transport. However, membrane topologies of AraH/RbsC
homologs were not readily predicted by a theoretical analysis since
overall hydrophobicities of their amino acid sequences are so broad as
to suggest five to eight putative TM helices (Hofmann and Stoffel
algorithm [11]). In order to determine the membrane structure of RbsC, we made a series of RbsC-PhoA fusions by using existing restriction enzyme sites, PCR mutagenesis, or exonucleolytic digestion (6, 20). The results indicate that the structure of RbsC comprises six TM helices and cytoplasmically located N and C termini.
RbsC-PhoA fusions generated specifically or randomly.
For
creating PhoA fusions at chosen sites, we used restriction enzymes (48N
and 316V) and PCR (93I, 117V, 120A, 123R, 126A, 129A, 152T, 199I, 264G,
313A, 317D, and 321Q). The sites were chosen to represent locations
which are likely to be in the hydrophilic regions based on the
hydropathy profile of the RbsC protein. The fusions were constructed in
the pYP110 plasmid, a derivative of pACYC184 which contains the
signal-sequence-less phoA gene (from the first amino acid of
the mature part) preceded by a multicloning site. The expression of
rbsC was driven by the promoter of the tetracycline
resistance gene from pACYC184. The fusions were introduced into the
BW14893 strain (27) lacking the phoA gene on the
chromosome, and their phenotypes were tested on indicator plates
containing 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine
(X-P).
To obtain random RbsC-PhoA fusions, the rbsC gene on the
plasmid (pYP113) was serially digested with exonuclease III and S1 nuclease and fused to phoA located on the same plasmid
(25). The pYP113 plasmid contains both genes in the same
orientation that allowed rejoining of the C-terminally deleted
rbsC to phoA which remained intact after
digestion with exonuclease III. The plasmid is an
rbsC-containing derivative of pYP110 in which
rbsC is expressed from the tetA promoter.
Linearization of pYP113 generated a 5' overhang downstream of
rbsC which was digested by exonuclease III (9).
Derivatives of pYP113 harboring the rbsC-phoA fusions were
then introduced into the BW14893 strain and grown on Luria-Bertani medium containing X-P. One hundred twenty-five blue colonies among 2,464 transformants were isolated, and their plasmids were analyzed by
restriction enzyme digestions. According to their sizes, the fusions
were classified into three groups, among which 60 were sequenced.
Fusions were found in 38 unique sites, which are located in the three
periplasmic regions of the proposed structure (Table 1 and Fig.
1).
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 1.
A proposed topology of RbsC. Residues in TM helices are
arranged in an  -helical structure. Circles and rectangles indicate
the residues of RbsC fused to PhoA. The periplasmic (P) and cytoplasmic
(C) loops are designated. The charged residues are indicated with plus
and minus signs.
|
|
For obtaining fusions in the cytoplasmic region, we first screened
white and pale blue colonies that showed positive responses to PhoA
immunoblotting, in order to avoid any out-of-frame construct. A total
of 18 plasmids were isolated and subjected to DNA sequencing. Sixteen
of them were found to be in frame with the phoA gene, giving
rise to 12 fusions with unique sites (Table 1). These sites are located
in the two cytoplasmic loops and the N- and C-terminal regions exposed
to the cytoplasm (Fig. 1).
PhoA activities and stabilities of the fusion proteins.
Alkaline phosphatase activities of the RbsC-PhoA fusions shown in Table
1 ranged from 1.1 to 355 U. They are divided into two groups based on
an arbitrary cutoff value of 75 U that can be used to predict their
locations, i.e., more than 75 for periplasmic and less than 75 for
cytoplasmic (8). As indicated in Fig. 1, it is likely that
RbsC has six TM helices with three periplasmic and two cytoplasmic
loops. Therefore, the N- and C-terminal regions of RbsC are located in
the cytoplasm. Almost all the alkaline phosphatase fusions agreed with
the six-TM model of RbsC. A possibility of RbsC having eight
transmembrane regions, in which either CI or PIII forms an extra pair
of TM peptides, has been carefully evaluated. However, it was deemed
unlikely considering that the lengths of amino acids for the loops are
barely enough to cover two TM spans and that the phoA
fusions in the middle of the CI and PIII loops have distinctly low and
high enzyme activities, respectively.
When steady-state levels of all the fusion products were analyzed by
immunoblotting from whole-cell extracts, proteins corresponding to the
RbsC-PhoA fusions and their degraded products were detected (only a few
are shown in Fig. 2) with various
intermediates and the PhoA moiety (47 kDa). In general, cytoplasmic
fusions with lower PhoA activities exhibited reduced levels of intact
or degraded PhoA proteins relative to periplasmic fusions (Fig. 2).
This might be due to the fact that alkaline phosphatase located in the
cytoplasm is often unable to fold correctly or to make its structure
stable (20). The low alkaline phosphatase activity (Table 1)
of the 264G fusion, predicted to be in a periplasmic loop, may be due to the instability of the fusion protein (6, 20). Indeed, the level of the protein was found (Fig. 2 and data not shown) to be
lower than that of the other periplasmic fusions. On the other hand,
the fusions in the C-terminal region, including 313A, 316V, 317D, 320K,
and 321Q, produced fairly stable PhoA products (Fig. 2 and data not
shown) despite their low alkaline phosphatase activities (ranging from
8.75 to 74.3 U). The reason might be that they have most of the
membrane peptides without any truncated TM regions. It is noted that
the fusions to inwardly directed TM peptides tended to exhibit high
alkaline phosphatase activities (Fig. 1), which might be due to the
fact that such fusions may reside in the periplasm (2).
View larger version (52K):
[in this window]
[in a new window]
|
FIG. 2.
Immunoblotting of RbsC-PhoA fusions. Equal amounts of
solubilized cells containing RbsC-PhoA fusion proteins were resolved by
sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis and
immunoblotted with anti-alkaline phosphatase antiserum. Activities of
alkaline phosphatase are designated underneath as more than 75 U ( )
or less than 75 U ( ). The positions of molecular mass standards
(kilodaltons) are indicated.
|
|
Localization of the C terminus by carboxypeptidase A
digestion.
The location of the C terminus of RbsC was further
confirmed by proteolysis. We constructed RbsC with a C-terminal His
tag, which was functional when tested for sugar uptake and utilization (data not shown). The protein was expressed in cells under the IPTG
(isopropyl-
-D-thiogalactopyranoside)-controlled
promoter, and right-side-out vesicles and inside-out vesicles (IOV)
were prepared as described elsewhere (13, 18). When
right-side-out vesicles and IOV that contained the RbsC-His-tagged
proteins were treated with carboxypeptidase A, the removal of the
C-terminal histidines was observed only in IOV, confirming the location
of the C-terminal region of RbsC in the cytoplasm (Fig.
3). However, the digestion was not
complete even after treatment for 90 min. This appears to be due to an
inaccessibility of the region to the enzyme, as reported for the
N-terminal region of HisQ, which was not effectively digested by
aminopeptidase (13). A similar approach employing
proteolysis with vesicles or spheroplasts has proven useful in
determining the topologies of ProW (7), HisQ, and HisM
(13). The topology of OppB was confirmed by trypsin treatment for permeable and impermeable cytoplasmic membranes (17).
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 3.
Carboxypeptidase A digestion of the C-terminal region of
RbsC. Right-side-out vesicles and IOV with RbsC-His-tagged proteins
were digested with carboxypeptidase A for various times (30, 60, and 90 min). The digestion products of vesicles were resolved by sodium
dodecyl sulfate-12% polyacrylamide gel electrophoresis and
immunoblotted with His tag conjugate. The immunoreactive bands were
quantified with the ImageQuant program. The white and black bars
indicate the percentages of the remaining immunoreactive bands of
right-side-out vesicles and IOV, respectively.
|
|
Topology of RbsC and its implication for other AraH family
members.
Our results suggest that RbsC is composed of six TM
helices (TM I, II, III, IV, V, and VI), each with a size of 20 to 22 aa (Fig. 1). Two TM regions (TM I and IV) possess only neutral amino acids, while TM II, III, V, and VI each contain a charged amino acid
(73D, 138R, 238E, and 306K, respectively). RbsC has three periplasmic
loops, I (13 aa), II (23 aa), and III (54 aa). The cytoplasmic loops I
(48 aa) and II (30 aa) also have +1 and +4 net charges, respectively
(Fig. 1). The presence of the EAA-G motif in the cytoplasmic loop II is
particularly notable, as it is conserved in all the membrane components
of the ABC transporters and is supposed to interact with the
cytoplasmic ATPase (RbsA [15]).
The N-terminal region of RbsC with a net charge of +3 (Fig. 1) is
predicted to be located in the cytoplasm, which is consistent with the
positive-inside rule (26), although the fusion at 2T with
low alkaline phosphatase activity (9.93 U) may not be sufficient to
prove its cytoplasmic location. The RbsC portion of the fusion is too
short to contain a topogenic signal while the protein itself is stable,
as seen on Western blots (data not shown). As a matter of fact, the
evidence for the cytoplasmic location of the N terminus was already
presented by the functionality of the RbsA-RbsC fusion in which the C
terminus of the cytoplasmic RbsA is connected to the N terminus of RbsC
(28). The short C-terminal region of RbsC has a net charge
of +1 (Fig. 1), as is also predicted for AlsC (for allose), AraH (for
arabinose), and MglC (for methyl-galactoside, all belonging to the AraH
family) with positive net charges in the C-terminal regions.
As predicted for RbsC, other hydrophobic components belonging to the
AraH superfamily may have six TM helices. In order to examine the
structures of some AraH members that are close to RbsC, we made
alkaline phosphatase fusions to the C-terminal regions of AlsC, AraH,
MglC, and XylH. They exhibited low alkaline phosphatase activities
(ranging from 2.47 to 24.45 U) with stable PhoA products (data not
shown). Therefore, it is very likely that these and other AraH family
members have structures very similar to that of RbsC. Some family
members with unknown functions include YphD, YjfF, YtfT, YdeY, and YdeZ
(from E. coli); TeuC1 and TeuC2 (from Rhizobium
tropici); GguB (from Agrobacterium tumefaciens); and Y4mJ (from Rhizobium sp. strain NGR234), which are putative
ABC-type sugar permeases with similarities to RbsC ranging from 22.8 to 33.5%. However, MalF, MalG, HisM, HisQ, OppB, OppC, and ProW in other
families are more distant from RbsC, with 8.6 to 14.4% similarity. The
majority of the inner membrane transport components other than the AraH
members, are predicted to have six membrane-spanning segments with
their amino and carboxy termini located in the cytoplasm (10).
In addition to their structural similarity, the AraH family members
appear to have functional similarity. For example, it was found that
the allose and xylose permeases can serve as a low-affinity transporter
for ribose (14, 24), indicating that these hydrophobic
components have substrate channels similar to those of RbsC.
Interestingly, the ydeY-ydeZ (E. coli) and
teuC1-teuC2 (R. tropici) pairs are each located
in one operon. It seems plausible that they form heterodimers to serve
as a functional permease, suggesting the possibility that RbsC forms a homodimer.
| |
ACKNOWLEDGMENTS |
We thank W. Goebel for anti-PhoA antiserum, B. L. Wanner for
the strain, H. Oh and S. Shin for the plasmids, and K. Park for computer analysis.
This work was supported by a grant, KOSEF 96-0401-01-01-3, from the
Korea Science and Engineering Foundation and in part by the Creative
Research Initiative Program.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: National
Creative Research Initiative Center for Behavioral Genetics, Department
of Biological Sciences, Korea Advanced Institute of Science and
Technology, Yusong-Ku, Taejon, Republic of Korea. Phone:
82-42-869-2629. Fax: 82-42-869-2610. E-mail:
ckpark{at}hanbit.kaist.ac.kr.
| |
REFERENCES |
| 1.
|
Bell, A. W.,
S. D. Buckel,
J. M. Groarke,
J. N. Hope,
D. H. Kingsley, and M. A. Hermodson.
1986.
The nucleotide sequences of the rbsD, rbsA, and rbsC genes of Escherichia coli K12.
J. Biol. Chem.
261:7652-7658[Abstract/Free Full Text].
|
| 2.
|
Boyd, D., and J. Beckwith.
1989.
Positively charged amino acid residues can act as topogenic determinants in membrane proteins.
Proc. Natl. Acad. Sci. USA
86:9446-9450[Abstract/Free Full Text].
|
| 3.
|
Boyd, D.,
C. Manoil, and J. Beckwith.
1987.
Determinants of membrane protein topology.
Proc. Natl. Acad. Sci. USA
84:8525-8529[Abstract/Free Full Text].
|
| 4.
|
Brickman, E., and J. Beckwith.
1975.
Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and  80 transducing phages.
J. Mol. Biol.
96:307-316[Medline].
|
| 5.
|
Dassa, E., and S. Muir.
1993.
Membrane topology of MalG, an inner membrane protein from the maltose transport system of Escherichia coli.
Mol. Microbiol.
7:29-38[Medline].
|
| 6.
|
Ginn, S. L.,
M. H. Brown, and R. A. Skurray.
1997.
Membrane topology of the metal-tetracycline/H+ antiporter TetA(K) from Staphylococcus aureus.
J. Bacteriol.
179:3786-3789[Abstract/Free Full Text].
|
| 7.
|
Haardt, M., and E. Bremer.
1996.
Use of phoA and lacZ fusions to study the membrane topology of ProW, a component of the osmoregulated ProU transport system of Escherichia coli.
J. Bacteriol.
178:5370-5381[Abstract/Free Full Text].
|
| 8.
|
Hagting, A.,
J. Velde,
B. Poolman, and W. N. Konings.
1997.
Membrane topology of the di- and tripeptide transport protein of Lactococcus lactis.
Biochemistry
36:6777-6785[Medline].
|
| 9.
|
Henikoff, S.
1984.
Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.
Gene
28:351-359[Medline].
|
| 10.
|
Higgins, C. F.
1992.
ABC transporters: from microorganism to man.
Annu. Rev. Cell Biol.
8:67-113.
|
| 11.
|
Hofmann, K., and W. Stoffel.
1993.
TM base a database of membrane spanning proteins segments.
Biol. Chem. Hoppe-Seyler
374:166-170.
|
| 12.
|
Hogg, R. W.,
C. Voelker, and I. Carlowitz.
1991.
Nucleotide sequence and analysis of the mgl operon of Escherichia coli K12.
Mol. Gen. Genet.
229:453-459[Medline].
|
| 13.
|
Kerppola, R. E., and G. F. Ames.
1992.
Topology of the hydrophobic membrane-bound components of the histidine periplasmic permease: comparison with other members of the family.
J. Biol. Chem.
267:2329-2336[Abstract/Free Full Text].
|
| 14.
|
Kim, C.,
S. Song, and C. Park.
1997.
The D-allose operon of Escherichia coli K-12.
J. Bacteriol.
179:7631-7637[Abstract/Free Full Text].
|
| 15.
|
Mourez, M.,
M. Hofnung, and E. Dassa.
1997.
Subunit interactions in ABC transporters: a conserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits.
EMBO J.
16:3066-3077[Medline].
|
| 16.
|
Mowbray, S. L.
1992.
Ribose and glucose-galactose receptors: competitors in bacterial chemotaxis.
J. Mol. Biol.
227:418-440[Medline].
|
| 17.
|
Pearce, S. R.,
M. L. Mimmack,
M. P. Gallagher,
U. Gileadi,
S. C. Hyde, and C. F. Higgins.
1992.
Membrane topology of the integral membrane components, OppB and OppC, of the oligopeptide permease of Salmonella typhimurium.
Mol. Microbiol.
6:47-57[Medline].
|
| 18.
|
Prossnitz, E.,
A. Gee, and G. F. Ames.
1989.
Reconstitution of the histidine periplasmic transport system in membrane vesicles: energy coupling and interaction between the binding protein and the membrane complex.
J. Biol. Chem.
264:5006-5014[Abstract/Free Full Text].
|
| 19.
|
Rosenfeld, S. A.,
P. E. Tevis, and N. W. Y. Ho.
1984.
Cloning and characterization of the xyl genes from Escherichia coli.
Mol. Gen. Genet.
194:410-415[Medline].
|
| 20.
|
Sarsero, J. P., and A. J. Pittard.
1995.
Membrane topology analysis of Escherichia coli K-12 Mtr permease by alkaline phosphatase and -galactosidase fusions.
J. Bacteriol.
177:297-306[Abstract/Free Full Text].
|
| 21.
|
Saurin, W., and E. Dassa.
1994.
Sequence relationships between integral inner membrane proteins of binding protein-dependent transport system: evolution by recurrent gene duplications.
Protein Sci.
3:325-344[Abstract].
|
| 22.
|
Saurin, W.,
W. Koster, and E. Dassa.
1994.
Bacterial binding protein-dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins.
Mol. Microbiol.
12:993-1004[Medline].
|
| 23.
|
Scripture, J. B.,
C. Voelker,
S. Miller,
R. T. O'Donnell,
L. Polgar,
J. Rade,
B. F. Horazdovsky, and R. W. Hogg.
1987.
High-affinity L-arabinose transport operon: nucleotide sequence and analysis of gene products.
J. Mol. Biol.
197:37-46[Medline].
|
| 24.
|
Song, S., and C. Park.
1998.
Utilization of D-ribose through D-xylose transporter.
FEMS Microbiol. Lett.
163:255-261[Medline].
|
| 25.
|
Sugiyama, J. E.,
S. Mahmoodian, and G. R. Jacobson.
1991.
Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions.
Proc. Natl. Acad. Sci. USA
88:9603-9607[Abstract/Free Full Text].
|
| 26.
|
von Heijne, G.
1986.
The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology.
EMBO J.
5:3021-3027[Medline].
|
| 27.
|
Wilmes-Riesenberg, M. R., and B. L. Wanner.
1992.
TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization.
J. Bacteriol.
174:4558-4575[Abstract/Free Full Text].
|
| 28.
|
Zaitseva, J.,
H. Zhang,
R. A. Binnie, and M. Hermodson.
1996.
The proteins encoded by the rbs operon of Escherichia coli. II. Use of chimeric protein constructs to isolate and characterize RbsC.
Protein Sci.
5:1100-1107[Abstract].
|
Journal of Bacteriology, February 1999, p. 1039-1042, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Tomasinsig, L., Scocchi, M., Mettulio, R., Zanetti, M.
(2004). Genome-Wide Transcriptional Profiling of the Escherichia coli Response to a Proline-Rich Antimicrobial Peptide. Antimicrob. Agents Chemother.
48: 3260-3267
[Abstract]
[Full Text]
-
Stewart, J. B., Hermodson, M. A.
(2003). Topology of RbsC, the Membrane Component of the Escherichia coli Ribose Transporter. J. Bacteriol.
185: 5234-5239
[Abstract]
[Full Text]
-
Ouchane, S., Kaplan, S.
(1999). Topological Analysis of the Membrane-localized Redox-responsive Sensor Kinase PrrB from Rhodobacter sphaeroides 2.4.1. J. Biol. Chem.
274: 17290-17296
[Abstract]
[Full Text]
Top
Abstract
Text
