To understand the structure of membrane transport proteins and their orientation across the membrane is necessary for determining the
molecular mechanism responsible for the phenomenon in a membrane transport system. There are many techniques available for the membrane topology determination, including chemical labeling, immunodetection, in vitro transcription/translation, and reporter gene fusions. In this thesis study, the structure of the CadA protein was examined experimentally using the genetic approach to construct various cadA gene fusions with the genes encoding either alkaline phosphatase (phoA) or β-galactosidase (lacZ) enzymes at different locations of the protein. This approach of using gene fusions to elucidate the architecture of membrane proteins has been applied in many protein topographical studies (Manoil, 1988 and 1991; Lee & Manoil, 1996).
Two reporter gene fusions were used in this study separately. The results of the two types of fusions were all consistent to each other and were used to propose the topological model as shown in Fig. 7. In this model, 8 transmembrane segments (TMs) were identified, along with 3 cytoplasmic loops, and 4 periplasmic loops were also found. In this model, the N-terminal domain of approximately 100 amino acid residues and the C-terminal domain of CadA protein were shown to be located in the cytoplasmic side. Taken together with the hydropathy data (using SOAP and TMpred method to predict) and positive-inside rule (von Heijne, 1992), we proposed that the TMs of CadA protein should be amended at the amino acid positions ranging from 105-123 (TM1),
131-151 (TM2), 164-192 (TM3+TM4), 332-356 (TM5), 363-391 (TM6),
677-697 (TM7), and 699-719 (TM8).
On the other hand, our data have demonstrated a difference in TM3 and TM4 when compared it to that of the Helicobacter CadA (Melchers et al., 1996, 1999). For example, the amino acid sequence of 164-192, including about 30 amino acids was originally predicted to be as a low hydrophobic domain, and seems unable to traverse the lipid bilayer by separating into two independent TMs (Fig. 2). Our data have shown that fusion enzyme activities on the fusion site E181, at the middle of the region, were asymmetric in this study. As we have predicted that enzyme activity of phoA fusions at E181, pE181P fusion, showed a high alkaline phosphatase activity (Table IVa), suggesting that the amino acid is at the periplasmic side as that of Helicobacter CadA (Melchers et al., 1999).
However, using lacZ fusion at the position, the pE181L clone, exhibited an unexpected high β-galactosidase activity which result disagrees with the data from phoA fusion at the same position and the Helicobacter CadA (Melchers et al., 1999; Table IV). The difference could be
explained that the periplasm-tended alkaline phosphatase is much easily to translocate the fusion site, E181, out to the periplasm; however, the larger cytoplasm-tended β-galactosidase would be kept in its cytoplasmic location due to the short hydrophobic region between the amino acid positions of 164 and 181 can not make the enzyme translocation.
Therefore, we believe that the region including the amino acid sequences from 164 to 192 are associated with the cytoplasmic membrane, but not traverses across the membrane. There is another possible explanation that the region is arranged around the hydrophilic channel of the transporter,
so it can be separated into two TMs in spite of its low hydrophobicity.
In general, our CadA topological results are similar to that of the
51
previously reported Helicobacter pylori homologous (Melchers et al., 1996, 1999). In these two CadA models, we found that both CadAs include 8 TMs and spanning on the membrane in a similar distribution of those common motifs (Fig. 8b). The conserved phosphatase domain (TGES motif) special to P-type ATPases is located between TM4 and TM5, while the signatures of phosphorylation (DKTGT motif) and ATP binding (GDGXNDXP motif) are positioned between TM6 and TM7. The conserved CPC motif found in all CPx-type ATPases is located within the TM6, as it was previously predicted (Solioz and Vulpe, 1996) and has demonstrated by the Helicobacterial CadA (Melchers et al., 1996, 1999).
On the other hand, there are some differences between these two CadAs. The staphylococcal CadA differs from the Helicobacter CadA in one of its highly hydrophobic regions between amino acid positions 626 and 646 in Staphylococcal CadA, but not found in its Helicobacter counterpart. This region was predicted as a putative TM by computer analysis but has not found in Helicobacter CadA (Fig. 2). Although our data did not support the presence of this putative TM, this hydrophobic region, on the other hand, might associate to the membrane and
participate the CadA function. Another difference is the high hydrophilic region found in the N-terminal of CadA protein, just before the TM1 and between amino acid positions 81 and 102. Within this region, the
staphylococcal CadA contains more charged amino acids than that of Helicobacter CadA (Fig. 4). The differences in the arrangement of the charged amino acids were found in other regions of these two enzymes.
For example, there are two lysine residues found in TM3 in
staphylococcal CadA, however, no lysine or other positively charged amino acid are found in this domain of Helicobacter CadA. Four
positively and one negatively charged amino acids are found in C-terminal end of staphylococcal CadA while only two positively charged amino acids are found in that of Helicobacter CadA.
An unexpected result was found in this topological study, that is, the neither chimeras G388P nor G388L displayed fusion enzyme activity (Table IV). The possible reason for that may be due to the circumstance that the reporter enzymes were fused immediately downstream of the TM VI. It was known that phoA and lacZ fusions tend to introduce biases into membrane protein topology analysis, especially when positively charged residues in the amino acid sequence downstream of the TM are absent in the fusion protein (Frank et al., 1999). Similarly, the reporter enzymes in these two clones were possibly embedded into the membrane because the lysine anchor at the amino acid position 392 was absent in these chimeras.
Putting all these information together, a detail CadA topography is thus to establish (Fig. 8).
Furthermore, we have demonstrated the presence of these chimera proteins in our study. Since there might be a possibility that no enzyme activity measured is due to no production in these chimera, therefore, it will be very important to clarify if these chimera proteins are produced in the study. As shown in Fig. 9, proteins prepared from those chimera clones were subject to SDS-PAGE analysis and the bands of the target proteins were visualized by using immunoblotting analysis.
Demonstrating that all these chimera proteins were produced in each particular clones, however, some with a small amount of protein
production, and some are larger (Fig. 9). It was noted that the periplasmic fusions with these reporter enzymes expressed less hybrid proteins than those of cytoplasmic fusions. This observation has also been previously
53
reported when the fusions with higher alkaline phosphatase activities are sometime toxic to cells, especially the fusions at the third periplasmic location (Allard and Bertrand, 1992). Similarly, the chimera protein with β-galactosidase is also toxic when secreted to the periplasm of E. coli (Snyder and Silhavy, 1995). Some other studies shown that
transmembrane-directed proteolysis of a membrane-spanning fusion protein causes periplasmic cleavage by a bacteriophage assembly protein (Guy-Caffey and Webster, 1993), a newly finding of a proteolytic
transmembrane signaling pathway, the β-lactamase regulatory system, might be also involved in the periplasmic cleavage (Zhang et al., 2001).
Therefore, it would not be surprised that these periplasmic fusions generate proteins slowly, but degrade them rapidly.
In some of these chimera proteins, bands of lower molecular weights were observed and these might be due to the degradation of these fusion proteins. As it has been suggested that an E. coli FtsH could
initiates proteolysis reaction to rapidly degrade fusion proteins if they were not tightly folded (Kihara et al., 1999). Therefore, along with those predicted fusion protein bands can be seen by Western blotting analysis, some degraded protein bands in each chimera sample preparation were also observed (Fig. 9). These data strongly demonstrated that all the chimera protein were made and their unable to detect either alkaline phosphatase or β-galactosidase enzyme activity in certain constructs were not due to the problem of protein production, but the enzyme orientation on the membrane instead. Among the chimera proteins analyzed in this study, we found that proteins produced by L130P, E181P and L109L clones displayed a higher molecular weight than they should be expected (Fig. 9). This latter observation could be explained as that the chimera
proteins maintaining native conformations and binding less SDS in the electrophoresis buffer as previously described (Guan et al., 1999). The mobility retardation might also result from the formation of disulfide bonds between the alkaline phosphatase (Derman and Beckwith, 1991).
As mentioned previously, more detail characterizations would be necessary to provide a close look into the common signatures in
CadA-related CPx-type ATPases, which including the metal binding property,phosphatase activity and aspartyl kinase activity of the enzyme.
Based on the hydropathy analysis (Fig. 2), and the membrane topological model of CadA presented here (Fig. 8), we discover some other features within the CadA protein and these structural specialties might be
important for CadA functions. For examples, does the highly charged C-terminal end of this protein represent a functional domain? Is the high hydrophobic region behind the putative ATP binding site significant for the function? To understand the exact mechanism involving these charged amino acids and other structures of CadA for its activity will require more data to put into the puzzle.
55
References
Aguiar JM. Guzman E. Martinez JL. Heavy metals, chlorine and antibiotic resistance in Escherichia coli isolates from ambulatory patients. Journal of Chemotherapy. 2(4):238-40, 1990
Allard JD. Bertrand KP. Membrane topology of the pBR322 tetracycline resistance protein. TetA-phoA gene fusions and implications for the mechanism of TetA membrane insertion. The Journal of Biological Chemistry. 267(25):17809-19, 1992
Bamberg K. and Sachs G. Topological analysis of H+,K(+)-ATPase using in vitro translation. The Journal of Biological Chemistry. 269:
16909-16919. 1994
Baranano DE. Wolosker H. Bae BI. Barrow RK. Snyder SH. Ferris CD. A mammalian iron ATPase induced by iron. The Journal of Biological Chemistry. 275(20):15166-73, 2000
Bayle D. Wangler S. Weitzenegger T. Steinhilber W. Volz J. Przybylski M.
Schafer KP. Sachs G. Melchers K. Properties of the P-type ATPases encoded by the copAP operons of Helicobacter pylori and
Helicobacter felis. Journal of Bacteriology. 180(2):317-29, 1998 Beard SJ. Hashim R. Membrillo-Hernandez J. Hughes MN. Poole RK.
Zinc(II) tolerance in Escherichia coli K-12: evidence that the zntA gene (o732) encodes a cation transport ATPase. Molecular
Microbiology. 25(5):883-91, 1997
Binet MR. Poole RK. Cd(II), Pb(II) and Zn(II) ions regulate expression of the metal-transporting P-type ATPase ZntA in Escherichia coli. FEBS Letters. 473(1):67-70, 2000
Bull PC. Thomas GR. Rommens JM. Forbes JR. Cox DW. The Wilson disease gene is a putative copper transporting P-type ATPase similar to
the Menkes gene [published erratum appears in Nat Genet 1994 Feb;6(2):214]. Nature Genetics. 5(4):327-37, 1993
Chaouni LB. Etienne J. Greenland T. Vandenesch F. Nucleic acid sequence and affiliation of pLUG10, a novel cadmium resistance plasmid from Staphylococcus lugdunensis. Plasmid. 36(1):1-8, 1996 Chang CN. Kuang WJ. and Chen EY. Nucleotide sequence of the alkaline
phosphatase gene of Escherichia coli. Gene 44 (1):121-125, 1986 Chelly J. Tumer Z. Tonnesen T. Petterson A. Ishikawa-Brush Y.
Tommerup N. Horn N. Monaco AP. Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein [see comments]. Nature Genetics. 3(1):14-9, 1993
Chikramane SG. Dubin DT. PsiTn554: A Staphylococcus aureus
chromosomal element encoding cadmium resistance determinants, and genes resembling the transposases genes of Tn554. Unpublished
Chopra I. Mechanism of plasmic-mediated resistance to cadmium in Staphylococcus aureus. Antimicrobial Agents & Chemotherapy.
7(1):8-14, 1975
Crupper SS. Worrell V. Stewart GC. Iandolo JJ. Cloning and expression of cadD, a new cadmium resistance gene of Staphylococcus aureus.
Journal of Bacteriology. 181(13):4071-5, 1999
Derman AI. Beckwith J. Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm. Journal of Bacteriology. 173(23):7719-22, 1991
Eckhardt M. Gotza B. Gerardy-Schahn R. Membrane topology of the mammalian CMP-sialic acid transporter. The Journal of Biological Chemistry. 274(13):8779-87, 1999
Ehrmann M. Bolek P. Mondigler M. Boyd D. Lange R. TnTIP and
57
TnTAP: mini-transposons for site-specific proteolysis in vivo.
Proceedings of the National Academy of Sciences of the United States of America. 94(24):13111-5, 1997
Ellis J. Carlin A. Steffes C. Wu J. Liu J. Rosen BP. Topological analysis of the lysine-specific permease of Escherichia coli. Microbiology. 141 ( Pt 8):1927-35, 1995
Engelman DM. Steitz TA. Goldman A. Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. [Review] [105 refs] Annual Review of Biophysics & Biophysical Chemistry.
15:321-53, 1986
Endo G. Silver S. CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258.
Journal of Bacteriology. 177(15):4437-41, 1995
Foster TJ. Plasmid-determined resistance to antimicrobial drugs and toxic metal ions in bacteria. [Review] [444 refs] Microbiological Reviews.
47(3):361-409, 1983
Fowler AV. Zabin I. Amino acid sequence of beta-galactosidase. XI.
Peptide ordering procedures and the complete sequence. The Journal of Biological Chemistry. 253 (15):5521-5525, 1978
Foye WO. Antimicrobial activities of mineral elements. In ED Weinberg, ed. Microorganisms and minerals. Microbiology series. Vol. 3. New York: Marcel Dekker, pp. 387, 1977
Franke CM. Tiemersma J. Venema G. Kok J. Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC.
Involvement of LcnC in lactococcin a maturation. The Journal of Biological Chemistry. 274(13):8484-90, 1999
Froshauer S. Green GN. Boyd D. McGovern K. Beckwith J. Genetic
analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli. Journal of Molecular Biology. 200(3):501-11, 1988
Fulkerson JF Jr. Mobley HL. Membrane topology of the NixA nickel transporter of Helicobacter pylori: two nickel transport-specific motifs within transmembrane helices II and III. Journal of Bacteriology.
182(6):1722-30, 2000
Guan L. Ehrmann M. Yoneyama H. Nakae T. Membrane topology of the xenobiotic-exporting subunit, mexB, of the mexA,B-oprM extrusion pump in Pseudomonas aeruginosa. The Journal of Biological
Chemistry 274(15):10517-10522, 1999
Guy-Caffey JK. Webster RE. The membrane domain of a bacteriophage assembly protein. Transmembrane-directed proteolysis of a
membrane-spanning fusion protein. The Journal of Biological Chemistry. 268(8):5488-95, 1993
Harlow E. and Lane D. Antibodies: A Laboratory Manual. 726 pages.
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988 Hirata T. Fujihira E. Kimura-Someya T. Yamaguchi A. Membrane
topology of the staphylococcal tetracycline efflux protein Tet(K) determined by antibacterial resistance gene fusion. Journal of Biochemistry. 124(6):1206-11, 1998
Hoffman DS. Wright A. Fusion of secreted proteins to alkaline
phosphatase: an approach for studying protein secretion. Proceedings of the National Academy of Sciences of the United States of America.
82:5107-11, 1985
Hofmann K. Stoffel W. TMbase - A database of membrane spanning proteins segments. Biological Chemistry Hoppe-Seyler 374:166. 1993
59
Holland EC. Drickamer K. Signal recognition particle mediates the insertion of a transmembrane protein which has a cytoplasmic NH2 terminus. The Journal of Biological Chemistry. 261(3):1286-92, 1986 Hustavova H. Obsitnikova K. Havranekova D. Influence of cadmium on
resistance to antibiotics in salmonellae isolated from pigs. Folia Microbiologica. 40(3):274-8, 1995
Inbar O. Ron EZ. Induction of cadmium tolerance in Escherichia coli K-12. FEMS Microbiology Letters. 113(2):197-200, 1993
Ivey DM. Guffanti AA. Shen Z. Kudyan N. Krulwich T.A. The cadC gene product of alkaliphilic Bacillus firmus OF4 partially restores Na+
resistance to an Escherichia coli strain lacking an Na+/H+ antiporter (NhaA). Journal of Bacteriology. 174 (15):4878-4884, 1992
Jennings ML. Anderson MP. Monaghan R. Monoclonal antibodies against human erythrocyte band 3 protein. Localization of proteolytic cleavage sites and stilbenedisulfonate-binding lysine residues. The Journal of Biological Chemistry. 261(19):9002-10, 1986
Jennings ML. Topography of membrane proteins. Annul Review of Biochemistry. 58:999-1027, 1989
Kanamaru K. Kashiwagi S. Mizuno T. The cyanobacterium,
Synechococcus sp. PCC7942, possesses two distinct genes encoding cation-transporting P-type ATPases. FEBS Letters. 330(1):99-104, 1993
Kihara A. Akiyama Y. Ito K. Dislocation of membrane proteins in FtsH-mediated proteolysis. EMBO Journal. 18(11):2970-81, 1999 Klein P. Kanehisa M. DeLisi C. The detection and classification of
membrane-spanning proteins. Biochimica et Biophysica Acta.
815(3):468-76, 1985
Knowles FC. Benson AA. The biochemistry of arsenic. Trends in Biochemical Sciences. 8: 178-180, 1983
Kyte J. Doolittle R. F. A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology. 157(1):105-32, 1982
LaRossa RA. Smulski DR. Van Dyk TK. Interaction of lead nitrate and cadmium chloride with Escherichia coli K-12 and Salmonella typhimurium global regulatory mutants. Journal of Industrial Microbiology. 14(3-4):252-8, 1995
Lebrun M. Audurier A. Cossart P. Plasmid-borne cadmium resistance genes in Listeria monocytogenes are similar to cadA and cadC of Staphylococcus aureus and are induced by cadmium. Journal of Bacteriology. 176 (10):3040-3048, 1994
Lee M. Manoil C. Molecular genetic analysis of membrane protein
topology. P. 189-2012. In Konings WN. Kaback HR. and Lolkema JS.
(ed.). Handbook of Biological Physics. Elsevier Science B. V. 1996 Lepke S. Passow H. Effects of incorporated trypsin on anion exchange
and membrane proteins in human red blood cell ghosts. Biochimica et Biophysica Acta. 455(2):353-70, 1976
Long JC. Wang S. Vik SB. Membrane topology of subunit a of the F1F0 ATP synthase as determined by labeling of unique cysteine residues.
The Journal of Biological Chemistry. 273(26):16235-40, 1998 Loo TW. Clarke DM. Covalent modification of human P-glycoprotein
mutants containing a single cysteine in either nucleotide-binding fold abolishes drug-stimulated ATPase activity. The Journal of Biological Chemistry. 270(39):22957-61, 1995
Lutsenko S. Kaplan JH. Organization of P-type ATPases: significance of
61
structural diversity. [Review] [54 refs] Biochemistry. 34(48):15607-13, 1995
MacLennan DH. Brandl CJ. Korczak B. Green NM. Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature. 316(6030):696-700, 1985
Manoil C. Analysis of membrane protein topology using alkaline
phosphatase and beta-galactosidase gene fusions. [Review] [27 refs]
Methods in Cell Biology. 34:61-75, 1991
Manoil C. Beckwith J. A genetic approach to analyzing membrane protein topology. Science. 233(4771):1403-8, 1986
Manoil C. Beckwith J. TnphoA: A transposon probe for protein export signals. Proceedings of the National Academy of Sciences of the United States of America. 82:8129-33, 1985
Manoil C. Boyd D. Beckwith J. Molecular genetic analysis of membrane protein topology. [Review] [39 refs] Trends in Genetics. 4(8):223-6, 1988
Manoil C. Mekalanos JJ. Beckwith J. Alkaline phosphatase fusions:
sensors of subcellular location. [Review] [48 refs] Journal of Bacteriology. 172(2):515-8, 1990
Melchers K. Schuhmacher A. Buhmann A. Weitzenegger T. Belin D.
Grau S. Ehrmann M. Membrane topology of CadA homologous P-type ATPase of Helicobacter pylori as determined by expression of phoA fusions in Escherichia coli and the positive inside rule. Research in Microbiology. 150(8):507-20, 1999
Melchers K. Weitzenegger T. Buhmann A. Steinhilber W. Sachs G.
Schafer KP. Cloning and membrane topology of a P type ATPase from
Helicobacter pylori. The Journal of Biological Chemistry. 271(1):
446-57, 1996
Mercer JF. Livingston J. Hall B. Paynter JA. Begy C. Chandrasekharappa S. Lockhart P. Grimes A. Bhave M. Siemieniak D. et al. Isolation of a partial candidate gene for Menkes disease by positional cloning [see comments]. Nature Genetics. 3(1):20-5, 1993
Michaelis S. Inouye H. Oliver D. Beckwith J. Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli. Journal of Bacteriology. 154(1):366-74, 1983
Miller JH. Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972
Nies D. Mergeay M. Friedrich B. Schlegel HG. Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes
eutrophus CH34. Journal of Bacteriology. 169(10):4865-8, 1987
Nies DH. Silver S. Plasmid-determined inducible efflux is responsible for resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus.
Journal of Bacteriology. 171(2):896-900, 1989
Nies DH. Nies A. Chu L. Silver S. Expression and nucleotide sequence of a plasmid-determined divalent cation efflux system from Alcaligenes eutrophus. Proceedings of the National Academy of Sciences of the United States of America. 86(19):7351-5, 1989
Nies DH. Resistance to cadmium, cobalt, zinc, and nickel in microbes.
[Review] [94 refs] Plasmid. 27(1):17-28, 1992
Novick RP. Murphy E. Gryczan TJ. Baron E. Edelman I. Penicillinase plasmids of Staphylococcus aureus: restriction-deletion maps. Plasmid.
2(1):109-29, 1979
Novick RP. Roth C. Plasmid-linked resistance to inorganic salts in
63
Staphylococcus aureus. Journal of Bacteriology. 95(4):1335-42, 1968 Nucifora G. Chu L. Misra TK. Silver S. Cadmium resistance from
Staphylococcus aureus plasmid pI258 cadA gene results from a cadmium-efflux ATPase. Proceedings of the National Academy of Sciences of the United States of America. 86(10):3544-8, 1989 Odermatt A. Suter H. Krapf R. Solioz M. An ATPase operon involved in
copper resistance by Enterococcus hirae. Annals of the New York Academy of Sciences. 671:484-6, 1992
Ogawa H. Haga T. Toyoshima C. Soluble P-type ATPase from an archaeon, Methanococcus jannaschii. FEBS Letters. 471(1):99-102, 2000
Ouchane S. Kaplan S. Topological analysis of the membrane-localized redox-responsive sensor kinase PrrB from Rhodobacter sphaeroides 2.4.1. The Journal of Biological Chemistry. 274(24):17290-96, 1999 Pedersen PL. Carafoli E. Ion motive ATPase. I. Ubiquity, properties, and
significance to cell function. Trends in Biochemical Sciences. 12:
146-150, 1987a
Pedersen P. L. Carafoli E. Ion motive ATPase. II. Eneergy coupling and work output. Trends in Biochemical Sciences. 12: 186-189, 1897b Perry RD. Silver S. Cadmium and manganese transport in Staphylococcus
aureus membrane vesicles. Journal of Bacteriology. 150(2):973-6, 1982
Phung L.T. Ajlani G. Haselkorn R. P-type ATPase from cyanobacterium
Phung L.T. Ajlani G. Haselkorn R. P-type ATPase from cyanobacterium