Evidence that KpsT, the ATP-binding component of an ATP-binding cassette transporter, is exposed to the periplasm and associates with polymer during translocation of the polysialic acid capsule of Escherichia coli K1

Bliss, Joseph M.; Silver, Richard P.

doi:10.1128/jb.179.4.1400-1403.1997

Cited by 24 publications

(20 citation statements)

References 25 publications

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“…This localization suggests intimate interaction between NBD1 and both TMDs. This is in agreement with present structural models that are based on studies on several ABC transporters in which NBD accessibility to proteases and biotinylated reagents from both sides of the membrane was investigated (4,6,20,49). These models propose that part of the NBDs may span the lipid bilayer and be exposed to the noncytoplasmic surface through the pore formed by the TMDs.…”

Section: Discussionsupporting

confidence: 75%

Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

et al. 2001

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The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrugassociated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V max of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.The yeast cadmium factor protein (Ycf1p) is a vacuolar membrane protein involved in heavy metal and drug detoxification in Saccharomyces cerevisiae. It is an ATP-dependent pump able to transport organic glutathione S (GS) conjugates (32), GS-metal complexes (18, 31), glutathione (41, 42), and other compounds, like unconjugated bilirubin (39). Ycf1p belongs to the ATP binding cassette (ABC) superfamily of transporters that includes the yeast a-factor transporter Ste6p (28), the Pfmdr-1 of Plasmodium falciparum which is associated with antimalarial drug resistance (17), and the human proteins P glycoprotein (16) and multidrug-associated protein (MRP1) (10) involved in multidrug resistance or the cystic fibrosis transmembrane conductance regulator, in which mutations cause cystic fibrosis (44). There are many sequence and mechanistic similarities between ABC transporters (21, 22), and they have a common evolutionary origin (11). Structural homology among ABC transporters reflects functional similarity in some cases, since MRP1 is able to suppress the Cd 2ϩ hypersensitivity of a yeast ⌬ycf1 mutant (57) and Ycf1p can transport the physiological substrate of MRP1, the leukotriene LTC 4 (15, 43). Ycf1p may thus be an excellent model for examining...

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Section: Discussionsupporting

confidence: 75%

Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

et al. 2001

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“…HisP is accessible to proteases and impermeant biotinylating reagents from both the cytoplasmic and the periplasmic sides of the membrane, indicating that it spans the lipid bilayer (22). This membrane-spanning property has also been demonstrated for other ABC transporters, such as MalK (23), the ATP-binding domain of CFTR (24,25), and KpsT (26). Because of the hydrophilic sequence of HisP, its membranespanning properties are likely to depend on a close interaction with the integral membrane subunits, HisQ and HisM (HisQM), with the strength of this interaction being important for regulating its ATPase activity.…”

mentioning

confidence: 74%

Modulation of ATPase Activity by Physical Disengagement of the ATP-binding Domains of an ABC Transporter, the Histidine Permease

Liu

Liu²,

Ames

1999

Journal of Biological Chemistry

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“…However, proteolytic digestion experiments using inverted and right-side-out membrane vesicles have revealed transmembrane topologies for the ATP-binding elements of the L-histidine transporter (HisP), the maltose transporter (MalK), and the polysialic acid transporter (KpsT) (36)(37)(38). Notably, increased proteolysis was observed with the E150G mutant of KpsT, which binds ATP and substrate but cannot hydrolyse the bound ATP and is thereby ''locked'' in a transmembrane state (38). This is similar to preprotein translocase studies using the analagous D209N SecA mutant, in which a blockage of ATP hydrolysis prevented the deinsertion of membrane-inserted SecA (30).…”

Section: Discussionmentioning

confidence: 99%

Both an N-terminal 65-kDa domain and a C-terminal 30-kDa domain of SecA cycle into the membrane at SecYEG during translocation

Eichler

Wickner

1997

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

103

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Evidence that KpsT, the ATP-binding component of an ATP-binding cassette transporter, is exposed to the periplasm and associates with polymer during translocation of the polysialic acid capsule of Escherichia coli K1

Cited by 24 publications

References 25 publications

Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

Modulation of ATPase Activity by Physical Disengagement of the ATP-binding Domains of an ABC Transporter, the Histidine Permease

Both an N-terminal 65-kDa domain and a C-terminal 30-kDa domain of SecA cycle into the membrane at SecYEG during translocation

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