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

Abstract

KpsT utilizes ATP to effect translocation of the polysialic acid capsule of Escherichia coli K1. We have previously proposed a mechanistic model for the action of this protein. Here, we provide evidence to support two predictions of the model: that KpsT associates with polymer and that KpsT is accessible from the periplasmic surface of the inner membrane.

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

“…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.…”

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

“…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.…”

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

“…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).…”