A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5

Abstract

The yeast ABC transporter Pdr5 plays a major role in drug resistance against a large number of structurally unrelated compounds. Although Pdr5 has been extensively studied, many important aspects regarding its molecular mechanisms remain unresolved. For example, a striking degeneration of conserved amino acid residues exists in the nucleotide binding domains (NBDs), but their functional relevance is unknown. Here, we performed in vivo and in vitro experiments to address the functional asymmetry of NBDs. It became evident by ATPase activity and drug transport studies that catalysis at only one of the two NBD composite sites is crucial for protein function. Furthermore, mutations of the proposed ''catalytic carboxylate'' (E1036) and the ''catalytic dyad histidine'' (H1068) were characterized. Although a mutation of the glutamate abolished ATPase activity and substrate transport, mutation of H1068 had no influence on ATP consumption. However, the H1068A mutation abolished rhodamine transport in vivo and in vitro, while leaving the transport of other substrates unaffected. By contrast to mammalian P-glycoprotein (P-gp), the ATPase activity of yeast Pdr5 is not stimulated by the addition of substrates, indicating that Pdr5 is an uncoupled ABC transporter that constantly hydrolyses ATP to ensure active substrate transport. Taken together, our data provide important insights into the molecular mechanism of Pdr5 and suggest that not solely the transmembrane domains dictate substrate selection.ATPase activity ͉ multidrug resistance ͉ substrate recognition

Generating Symmetry in the Asymmetric ATP-binding Cassette (ABC) Transporter Pdr5 from Saccharomyces cerevisiae

“…Although this degenerate site could bind ATP, hydrolysis should be impossible or severely impaired due to the absence of these key residues. Some initial mutational studies supported this view (10). This observation of a degenerate and a consensus site is common in yeast ABC transporters but is uncommon in ABC transporters of archaea and bacteria.…”

“…Cells were harvested at A 600 of 3.5. The isolation of plasma membranes was performed as described elsewhere (8,10).…”

“…Pdr5 displays a high basal ATPase activity, which, unlike its other human counterparts such as P-gp, does not seem to be stimulated in the presence of substrates (7,10). Such an uncoupling has important mechanistic consequences because ATP hydrolysis is supposed to be the crucial step that triggers the transport of substrates across the membrane, and in the proper environment, it is strictly coupled to substrate translocation (11).…”

Structure and mechanism of ATP-binding cassette transporters

“…Some type I ABC importers are tightly coupled and do not exhibit futile ATP hydrolysis, which made it possible to unequivocally determine the reaction stoichiometry as two ATPs per transported substrate (Patzlaff et al 2003). Notably, futile ATP hydrolysis has recently been observed in the yeast multi-drug ABC transporter Pdr5 (Ernst et al 2008). The relevant point for a productive transport cycle through type I ABC importers is that upon binding of ATP, a transient-binding pocket for substrate is formed within the translocation pathway, but is only accessible from the outside.…”

Evidence for a Molecular Diode-based Mechanism in a Multispecific ATP-binding cassette (ABC) Exporter

“…S1368A exhibited profound, broad multidrug hypersensitivity, and yet its ATPase activity was indistinguishable from WT, as was the allosteric inhibition (trans inhibition) of this enzyme by clo. Previously reported mutations in Pdr5 with broad hypersensitivity, such as K911A (Walker A), E1036Q, and G312A, exhibited either a strong reduction in steady-state ATPase activity (13,20) or a loss of communication between the transmembrane domains and NBDs causing a large reduction in allosteric inhibition (14,17). This includes the S1360F mutant, which is in the same TMH as S1368A (21,24).…”