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Journal of Bacteriology, August 2001, p. 4761-4770, Vol. 183, No. 16
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.16.4761-4770.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Juan M. Falcón-Pérez,1 Mónica Martínez-Burgos,1 Jesús Molano,2 María J. Mazón,1 and Pilar Eraso1,*

Instituto de Investigaciones Biomédicas "Alberto Sols," CSIC-UAM,1 and Unidad de Genética Molecular, Servicio de Bioquímica, Hospital La Paz,2 Madrid, Spain

Received 8 February 2001/Accepted 24 May 2001

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 multidrug-associated 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 Vmax 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.

* Corresponding author. Mailing address: Instituto de Investigaciones Biomédicas "Alberto Sols" CSIC-UAM, Arturo Duperier 4, 28029 Madrid, Spain. Phone: 34 91 585 4616. Fax: 34 91 585 4587. E-mail: peraso{at}iib.uam.es.

Journal of Bacteriology, August 2001, p. 4761-4770, Vol. 183, No. 16
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.16.4761-4770.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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