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Hajime Tokuda - One of the best experts on this subject based on the ideXlab platform.
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defective lipoprotein sorting induces Lola expression through the rcs stress response phosphorelay system
Journal of Bacteriology, 2012Co-Authors: Kazuyuki Tao, Shinichiro Narita, Hajime TokudaAbstract:The Escherichia coli Lola protein is a lipoprotein-specific chaperone that carries lipoproteins from the inner to the outer membrane. A dominant negative Lola mutant, Lola(I93C/F140C), in which both (93)Ile and (140)Phe are replaced by Cys, binds tightly to the lipoprotein-dedicated ABC transporter LolCDE complex on the inner membrane and therefore inhibits the detachment of outer membrane-specific lipoproteins from the inner membrane. We found that the expression of this mutant strongly induced Lola gene transcription. The depletion of the Lola or LolB protein also triggered Lola gene transcription, indicating that the inhibition of outer membrane lipoprotein transport triggers Lola transcription. To elucidate the mechanism, we isolated mutants that are unable to induce Lola transcription using the lacZ gene fused to the Lola promoter as a reporter and found that the Rcs phosphorelay system directly regulates Lola transcription. An outer membrane lipoprotein, RcsF, was essential for this activation, while the coactivator RcsA was dispensable. Taking the observation that an RcsF mutant localized in the inner membrane constitutively activated the Rcs phosphorelay system into consideration, the results shown here strongly suggest that correct lipoprotein sorting to the outer membrane is monitored by RcsF, which plays a key role in the Rcs stress response system.
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structural investigation of the interaction between Lola and lolb using nmr
Journal of Biological Chemistry, 2009Co-Authors: Shingo Nakada, Hajime Tokuda, Suguru Okuda, Masayoshi Sakakura, Hideo Takahashi, Ichio ShimadaAbstract:Lipoproteins that play critical roles in various cellular functions of Gram-negative bacteria are localized in the cells inner and outer membranes. Lol proteins (Lola, LolB, LolC, LolD, and LolE) are involved in the transportation of outer membrane-directed lipoproteins from the inner to the outer membrane. Lola is a periplasmic chaperone that transports lipoproteins, and LolB is an outer membrane receptor that accepts lipoproteins. To clarify the structural basis for the lipoprotein transfer from Lola to LolB, we examined the interaction between Lola and mLolB, a soluble mutant of LolB, using solution NMR spectroscopy. We determined the interaction mode between Lola and mLolB with conformational changes of Lola. Based upon the observations, we propose that the Lola·LolB complex forms a tunnel-like structure, where the hydrophobic insides of Lola and LolB are connected, which enables lipoproteins to transfer from Lola to LolB.
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model of mouth to mouth transfer of bacterial lipoproteins through inner membrane lolc periplasmic Lola and outer membrane lolb
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Suguru Okuda, Hajime TokudaAbstract:Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, Lola. Lola then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detail. However, it remained unclear how Lol factors interact with each other to conduct very efficient lipoprotein transfer in the periplasm where ATP is not available. To address this issue, a photo-reactive phenylalanine analogue, p-benzoyl-phenylalanine, was introduced at various positions of Lola and LolB, of which the overall structures are very similar and comprise an incomplete β-barrel with a hydrophobic cavity inside. Cells expressing Lola or LolB derivatives containing the above analogue were irradiated with UV for in vivo photo-cross-linking. These analyses revealed a hot area in the same region of Lola and LolB, through which Lola and LolB interact with each other. This area is located at the entrance of the hydrophobic cavity. Moreover, this area in Lola is involved in the interaction with a membrane subunit, LolC, whereas no cross-linking occurs between Lola and the other membrane subunit, LolE, or ATP-binding subunit LolD, despite the structural similarity between LolC and LolE. The hydrophobic cavities of Lola and LolB were both found to bind lipoproteins inside. These results indicate that the transfer of lipoproteins through Lol proteins occurs in a mouth-to-mouth manner.
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opening and closing of the hydrophobic cavity of Lola coupled to lipoprotein binding and release
Journal of Biological Chemistry, 2008Co-Authors: Yuki Oguchi, Shoji Watanabe, Naoko Yokota, Kazuki Takeda, Kunio Miki, Hajime TokudaAbstract:Outer membrane-specific lipoproteins of Escherichia coli are released from the inner membrane through the action of Lol-CDE, which leads to the formation of a complex between the lipoprotein and Lola, a periplasmic chaperone. Lola then transfers lipoproteins to LolB, a receptor in the outer membrane. The structures of Lola and LolB are very similar, having an incomplete β-barrel covered with an α-helical lid forming a hydrophobic cavity inside. The cavity of Lola, but not that of LolB, is closed and thus inaccessible to the bulk solvent. Previous studies suggested that Arg at position 43 of Lola is critical for maintaining this closed structure. We show here, through a crystallographic study, that the cavity of the Lola(R43L) mutant, in which Leu replaces Arg-43, is indeed open to the external milieu. We then found that the binding of a fluorescence probe distinguishes the open/close state of the cavity. Furthermore, it was revealed that the hydrophobic cavity of Lola opens upon the binding of lipoproteins. Such a liganded Lola was found to be inactive in the release of lipoproteins from the inner membrane. On the other hand, the liganded Lola became fully functional when lipoproteins were removed from Lola by detergent treatment or transferred to LolB. Free Lola thus formed was inaccessible to a fluorescence probe. These results, taken together, reveal the Lola cycle, in which the hydrophobic cavity undergoes opening and closing upon the binding and release of lipoproteins, respectively.
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a short helix in the c terminal region of Lola is important for the specific membrane localization of lipoproteins
FEBS Letters, 2008Co-Authors: Suguru Okuda, Shoji Watanabe, Hajime TokudaAbstract:The structures of a lipoprotein carrier, Lola, and a lipoprotein receptor, LolB, are similar except for an extra C-terminal loop containing a 310 helix and β-strand 12 in Lola. Lipoprotein release was significantly reduced when β-12 was deleted. Deletion of the 310 helix also inhibited the lipoprotein release. Furthermore, lipoproteins were non-specifically localized to membranes when Lola lacked the 310 helix. Thus, the membrane localization of lipoproteins with the Lola derivative lacking the 310 helix was independent of LolB whereas LolB was essential for the outer membrane localization of lipoproteins with the wild-type Lola.
Maldonado Orihuela, Esteban Jhordy - One of the best experts on this subject based on the ideXlab platform.
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Efecto in vitro de bebidas carbonatadas con edulcorantes caloricos y acaloricos sobre streptococcus mutans atcc 25175
Universidad Privada Antenor Orrego - UPAO, 2017Co-Authors: Maldonado Orihuela, Esteban JhordyAbstract:OBJECTIVE: To determine the in vitro effect of carbonated beverages with caloric and no calorics sweeteners on Streptococcus mutans ATCC 25175. MATERIALS AND METHODS: The prospective, longitudinal, comparative and experimental study. The sample consisted of 176 test tubes. Contents of macrodilution of carbonated beverages and Streptococcus mutans ATCC 25175 in Broth Heart Infusion (BHI). The effect of carbonated beverages on Streptococcus mutans ATCC 25175 was determined through the Minimum Inhibitory Concentration (MIC). Statistical analysis was performed using the ANOVA test that was completed with the DUNCAN test. RESULTS: Carbonated beverages with caloric sweeteners showed that in almost all the concentrations tested, the data obtained are much higher than the positive control; Finding the MIC at 50% for Coca Cola (original), while for the Inca Kola beverage (original), the MIC was found at 25%. In contrast, the (zero) presentation of carbonated beverages with no caloric sweeteners studied, Coca Cola and Inca Kola, the data obtained from the spectrophotometry were below the positive control, so it was not possible to find the MIC. CONCLUSION: Carbonated beverages with caloric sweeteners at most concentrations worked up stimulated the in vitro growth of Streptococcus mutans ATCC 25175. While for carbonated beverages with no caloric sweeteners, all concentrations studied decrease the in vitro growth of Streptococcus mutans ATCC 25175.OBJETIVO: Determinar el efecto in vitro de bebidas carbonatadas con edulcorantes calóricos y acalóricos sobre el Streptococcus mutans ATCC 25175. MATERIALES Y MÉTODOS: El estudio prospectivo, longitudinal, comparativo y experimental. La muestra estuvo constituida por 176 tubos de ensayo. Conteniendo la macrodilución de las bebidas carbonatadas y Streptococcus mutans ATCC 25175 en caldo Infusión cerebro corazón (BHI). El efecto de las bebidas carbonatas sobre el Streptococcus mutans ATCC 25175, se determinó a través de la Concentración Mínima Inhibitoria (CMI). El análisis estadístico se realizó mediante la prueba ANOVA que fue completada con la prueba de DUNCAN. RESULTADOS: Las bebidas carbonatadas con edulcorantes calóricos mostraron que en casi todas las concentraciones ensayadas, los datos obtenidos son muy superiores al control positivo; encontrando la CMI en el 50% para la Coca Cola (original), mientras que para la bebida Inca Kola (original), la CMI se halló en el 25%. Contrariamente, la presentación (zero) de las bebidas carbonatadas con edulcorantes acalóricos estudiados, Coca Cola e Inca Kola, los datos obtenidos de la espectrofotometría estuvieron por debajo del control positivo, por lo que no fue posible encontrar la CMI. CONCLUSIÓN: Las bebidas carbonatadas con edulcorantes calóricos, en la mayoría de las concentraciones trabajadas, estimulan al crecimiento in vitro del Streptococcus mutans ATCC 25175. Mientras que para las bebidas carbonatadas con edulcorantes acalóricos, todas las concentraciones estudiadas disminuyeron el crecimiento in vitro del Streptococcus mutans ATCC 25175
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Efecto in vitro de bebidas carbonatadas con edulcorantes calóricos y acalóricos sobre Streptococcus mutans ATCC 25175
Universidad Privada Antenor Orrego - UPAO, 2017Co-Authors: Maldonado Orihuela, Esteban JhordyAbstract:OBJETIVO: Determinar el efecto in vitro de bebidas carbonatadas con edulcorantes calóricos y acalóricos sobre el Streptococcus mutans ATCC 25175. MATERIALES Y MÉTODOS: El estudio prospectivo, longitudinal, comparativo y experimental. La muestra estuvo constituida por 176 tubos de ensayo. Conteniendo la macrodilución de las bebidas carbonatadas y Streptococcus mutans ATCC 25175 en caldo Infusión cerebro corazón (BHI). El efecto de las bebidas carbonatas sobre el Streptococcus mutans ATCC 25175, se determinó a través de la Concentración Mínima Inhibitoria (CMI). El análisis estadístico se realizó mediante la prueba ANOVA que fue completada con la prueba de DUNCAN. RESULTADOS: Las bebidas carbonatadas con edulcorantes calóricos mostraron que en casi todas las concentraciones ensayadas, los datos obtenidos son muy superiores al control positivo; encontrando la CMI en el 50% para la Coca Cola (original), mientras que para la bebida Inca Kola (original), la CMI se halló en el 25%. Contrariamente, la presentación (zero) de las bebidas carbonatadas con edulcorantes acalóricos estudiados, Coca Cola e Inca Kola, los datos obtenidos de la espectrofotometría estuvieron por debajo del control positivo, por lo que no fue posible encontrar la CMI. CONCLUSIÓN: Las bebidas carbonatadas con edulcorantes calóricos, en la mayoría de las concentraciones trabajadas, estimulan al crecimiento in vitro del Streptococcus mutans ATCC 25175. Mientras que para las bebidas carbonatadas con edulcorantes acalóricos, todas las concentraciones estudiadas disminuyeron el crecimiento in vitro del Streptococcus mutans ATCC 25175.OBJECTIVE: To determine the in vitro effect of carbonated beverages with caloric and no calorics sweeteners on Streptococcus mutans ATCC 25175. MATERIALS AND METHODS: The prospective, longitudinal, comparative and experimental study. The sample consisted of 176 test tubes. Contents of macrodilution of carbonated beverages and Streptococcus mutans ATCC 25175 in Broth Heart Infusion (BHI). The effect of carbonated beverages on Streptococcus mutans ATCC 25175 was determined through the Minimum Inhibitory Concentration (MIC). Statistical analysis was performed using the ANOVA test that was completed with the DUNCAN test. RESULTS: Carbonated beverages with caloric sweeteners showed that in almost all the concentrations tested, the data obtained are much higher than the positive control; Finding the MIC at 50% for Coca Cola (original), while for the Inca Kola beverage (original), the MIC was found at 25%. In contrast, the (zero) presentation of carbonated beverages with no caloric sweeteners studied, Coca Cola and Inca Kola, the data obtained from the spectrophotometry were below the positive control, so it was not possible to find the MIC. CONCLUSION: Carbonated beverages with caloric sweeteners at most concentrations worked up stimulated the in vitro growth of Streptococcus mutans ATCC 25175. While for carbonated beverages with no caloric sweeteners, all concentrations studied decrease the in vitro growth of Streptococcus mutans ATCC 25175.Tesi
Shinichi Matsuyama - One of the best experts on this subject based on the ideXlab platform.
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crystal structures of bacterial lipoprotein localization factors Lola and lolb
The EMBO Journal, 2003Co-Authors: Kazuki Takeda, Naoko Yokota, Hajime Tokuda, Hideyuki Miyatake, Shinichi Matsuyama, Kunio MikiAbstract:Lipoproteins having a lipid‐modified cysteine at the N‐terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram‐negative bacteria. We determined the crystal structures of a periplasmic chaperone, Lola, and an outer membrane lipoprotein receptor, LolB. Despite their dissimilar amino acid sequences, the structures of Lola and LolB are strikingly similar to each other. Both have a hydrophobic cavity consisting of an unclosed β barrel and an α‐helical lid. The cavity represents a possible binding site for the lipid moiety of lipoproteins. Detailed structural differences between the two proteins provide significant insights into the molecular mechanisms underlying the energy‐independent transfer of lipoproteins from Lola to LolB and from LolB to the outer membrane. Furthermore, the structures of both Lola and LolB determined from different crystal forms revealed the distinct structural dynamics regarding the association and dissociation of lipoproteins. The results are discussed in the context of the current model for the lipoprotein transfer from the inner to the outer membrane through a hydrophilic environment.
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dominant negative mutant of a lipoprotein specific molecular chaperone Lola tightly associates with lolcde
FEBS Letters, 2002Co-Authors: Atsuki Miyamoto, Shinichi Matsuyama, Hajime TokudaAbstract:Periplasmic molecular chaperone Lola and the inner membrane ATP binding cassette transporter LolCDE are essential for ATP-dependent release of outer membrane-directed lipoproteins from the inner membrane of Escherichia coli. A Lola(F47E) mutant carrying a Phe to Glu mutation at position 47 was defective in the release of lipoproteins from spheroplasts and proteoliposomes reconstituted with LolCDE. When incubated with proteoliposomes containing LolCDE, Lola remained in the supernatant whereas Lola(F47E) bound to proteoliposomes. This tight association of Lola(F47E) with LolCDE caused a dominant negative phenotype in vivo, suggesting that the Lola-LolCDE interaction is critical for lipoprotein release.
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mutant of Lola a lipoprotein specific molecular chaperone of escherichia coli defective in the transfer of lipoproteins to lolb
Biochemical and Biophysical Research Communications, 2001Co-Authors: Atsuki Miyamoto, Shinichi Matsuyama, Hajime TokudaAbstract:The outer membrane-specific lipoproteins of Escherichia coli are released from the inner membrane as a water-soluble complex with Lola and then transferred to the outer membrane receptor, LolB. Lola thus plays a critical role in the sorting and outer membrane localization of lipoproteins. To dissect the Lola function, the highly conserved residues were subjected to random mutagenesis, followed by selection for a growth defect. Lola(R43L), one of mutants thus constructed, possessed Leu in place of Arg at position 43 and caused accumulation of the Lola(R43L)-lipoprotein complex in the periplasm. Lola(R43L) was as active as wild-type Lola as to the release of lipoproteins from spheroplasts. In marked contrast, the transfer of lipoproteins from Lola(R43L) to LolB was completely inhibited, indicating that Arg at position 43 of Lola is involved in the lipoprotein transfer reaction.
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characterization of the Lola lolb system as the general lipoprotein localization mechanism of escherichia coli
Journal of Biological Chemistry, 1999Co-Authors: Naoko Yokota, Shinichi Matsuyama, Toshiro Kuroda, Hajime TokudaAbstract:The major outer membrane lipoprotein (Lpp) ofEscherichia coli requires Lola for its release from the cytoplasmic membrane, and LolB for its localization to the outer membrane. We examined the significance of the Lola-LolB system as to the outer membrane localization of other lipoproteins. All lipoproteins possessing an outer membrane-directed signal at the N-terminal second position were efficiently released from the inner membrane in the presence of Lola. Some lipoproteins were released in the absence of externally added Lola, albeit at a slower rate and to a lesser extent. This Lola-independent release was also strictly dependent on the outer membrane sorting signal. A lipoprotein-Lola complex was formed when the release took place in the presence of Lola, whereas lipoproteins released in the absence of Lola existed as heterogeneous complexes, suggesting that the release and the formation of a complex with Lola are distinct events. The release of LolB, an outer membrane lipoprotein functioning as the receptor for a lipoprotein-Lola complex, occurred with a trace amount of Lola, and therefore was extremely efficient. The Lola-dependent release of lipoproteins was found to be crucial for the specific incorporation of lipoproteins into the outer membrane, whereas lipoproteins released in the absence of Lola were nonspecifically and inefficiently incorporated into the membrane. The outer membrane incorporation of lipoproteins including LolB per se was dependent on LolB in the outer membrane. From these results, we conclude that lipoproteins in E. coli generally utilize the Lola-LolB system for efficient release from the inner membrane and specific localization to the outer membrane.
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genetic analyses of the in vivo function of Lola a periplasmic chaperone involved in the outer membrane localization of escherichia coli lipoproteins
FEBS Letters, 1998Co-Authors: Terutaka Tajima, Naoko Yokota, Shinichi Matsuyama, Hajime TokudaAbstract:The major outer membrane lipoprotein (Lpp) of Escherichia coli is released from the cytoplasmic membrane into the periplasm as a complex with Lola, a periplasmic chaperone, prior to the localization in the outer membrane. To determine whether or not Lola is generally involved in the outer membrane localization of lipoproteins in vivo, the chromosomal Lola gene was manipulated so as to be controlled by the lac promoter-operator. Depletion of Lola caused a severe growth defect, and impaired the outer membrane localization of Lpp and Pal, another outer membrane lipoprotein. Although Lola depletion did not immediately arrest the growth of cells lacking Lpp, disruption of the chromosomal Lola gene was lethal to the lpp strain, indicating that Lola is generally required for the outer membrane localization of lipoproteins, and therefore essential irrespective of the presence or absence of Lpp.
Azeddine Driouich - One of the best experts on this subject based on the ideXlab platform.
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characterisation of cell wall polysaccharides arabinogalactans proteins agps and phenolics of cola nitida cola acuminata and garcinia kola seeds
Carbohydrate Polymers, 2009Co-Authors: Thaddee Boudjeko, Christophe Rihouey, Denis Omokolo Ndoumou, Ismail El Hadrami, Patrice Lerouge, Azeddine DriouichAbstract:Abstract Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata , C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola . Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for ∼50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.
Atsuki Miyamoto - One of the best experts on this subject based on the ideXlab platform.
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dominant negative mutant of a lipoprotein specific molecular chaperone Lola tightly associates with lolcde
FEBS Letters, 2002Co-Authors: Atsuki Miyamoto, Shinichi Matsuyama, Hajime TokudaAbstract:Periplasmic molecular chaperone Lola and the inner membrane ATP binding cassette transporter LolCDE are essential for ATP-dependent release of outer membrane-directed lipoproteins from the inner membrane of Escherichia coli. A Lola(F47E) mutant carrying a Phe to Glu mutation at position 47 was defective in the release of lipoproteins from spheroplasts and proteoliposomes reconstituted with LolCDE. When incubated with proteoliposomes containing LolCDE, Lola remained in the supernatant whereas Lola(F47E) bound to proteoliposomes. This tight association of Lola(F47E) with LolCDE caused a dominant negative phenotype in vivo, suggesting that the Lola-LolCDE interaction is critical for lipoprotein release.
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mutant of Lola a lipoprotein specific molecular chaperone of escherichia coli defective in the transfer of lipoproteins to lolb
Biochemical and Biophysical Research Communications, 2001Co-Authors: Atsuki Miyamoto, Shinichi Matsuyama, Hajime TokudaAbstract:The outer membrane-specific lipoproteins of Escherichia coli are released from the inner membrane as a water-soluble complex with Lola and then transferred to the outer membrane receptor, LolB. Lola thus plays a critical role in the sorting and outer membrane localization of lipoproteins. To dissect the Lola function, the highly conserved residues were subjected to random mutagenesis, followed by selection for a growth defect. Lola(R43L), one of mutants thus constructed, possessed Leu in place of Arg at position 43 and caused accumulation of the Lola(R43L)-lipoprotein complex in the periplasm. Lola(R43L) was as active as wild-type Lola as to the release of lipoproteins from spheroplasts. In marked contrast, the transfer of lipoproteins from Lola(R43L) to LolB was completely inhibited, indicating that Arg at position 43 of Lola is involved in the lipoprotein transfer reaction.
