Identification of critical residues for transport activity of Acr3p, the Saccharomyces cerevisiae As(III)/H+ antiporter
Authors: Magdalena Migocka
Katarzyna Markowska
Robert Wysocki
Ewa Maciaszczyk-Dziubinska
Donata Wawrzycka
Journal: Molecular Microbiology (2015)
Discipline: MICROBIOLOGY
Keywords: Acr3p; antiporter; As(III); H(+); transmembrane segment
Affiliations: Institute of Experimental Biology, University of Wroclaw, 50-328, Wroclaw, Poland.

Abstract:  Acr3p is an As(III)/H(+) antiporter from Saccharomyces cerevisiae belonging to the bile/arsenite/riboflavin transporter superfamily. We have previously found that Cys151 located in the middle of the fourth transmembrane segment (TM4) is critical for antiport activity, suggesting that As(III) might interact with a thiol group during the translocation process. In order to identify functionally important residues involved in As(III)/H(+) exchange, we performed a systematic alanine-replacement analysis of charged/polar and aromatic residues that are conserved in the Acr3 family and located in putative transmembrane segments. Nine residues (Asn117, Trp130, Arg150, Trp158, Asn176, Arg230, Tyr290, Phe345, Asn351) were found to be critical for proper folding and trafficking of Acr3p to the plasma membrane. In addition, we found that replacement of highly conserved Phe266 (TM7), Phe352 (TM9), Glu353 (TM9) and Glu380 (TM10) with Ala abolished transport activity of Acr3p, while mutation of Ser349 (TM9) to Ala significantly reduced the As(III)/H(+) exchange, suggesting an important role of these residues in the transport mechanism. Detailed mutational analysis of Glu353 and Glu380 revealed that the negatively charged residues located in the middle of transmembrane segments TM9 and TM10 are crucial for antiport activity. We also discuss a hypothetical model of the Acr3p transport mechanism. 

Retracted: No

PubPeer comments:
 In Figure 4 the same set of control bands is used several times for different Acr3 mutants. ![file] , 
 Could the authors please comment on the status of this paper? , 
 Correction was issued: https: onlinelibrary.wiley.com doi 10.1111 mmi.14598 , 
 Quite a correction. u201cIn the above article, the authors compiled Figure 2 incorrectly by inadvertently using the same image of the growth of acr3 mutants on a control plate without metalloids for two set of transformants (from T261A to E380A), while the image of the control plate depicted the growth of transformants from T261A to T346A was used as the image showing the growth of transformants labeled as u201cvecu201d up to the W118A mutant. The correct images for Figure 2 are shown below. The authors also compiled Figure 4a incorrectly by using Western blots detecting Cdc28 Pho85 proteins levels as a loading control that did not match the corresponding Western blots depicting the total protein levels of mutated versions of Acr3u2010GFP. In addition, two images of loading controls were duplicated and one image was imprecisely cut out from the original blot. The correct images for Figure 4a are shown below. The authors also compiled Figure 6c incorrectly by using Western blots detecting Pma1 proteins levels as a loading control that did not match the corresponding Western blots depicting the total protein levels of mutated versions of Acr3u2010GFP (i.e., E380D, E380K, E380Q, E380L, and E380A). The correct images for Figure 6c are shown below. The authors sincerely apologize for these errors and emphasize that they do not affect the conclusions reported in the paper.u201d *** ![file] *** ![file] *** ![file] ,