Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2
Authors: Magdalena Migocka
Anna Kosieradzaka
Anna Papierniak
Ewelina Posyniak
Ewa Maciaszczyk-Dziubinska
Journal: Journal of Biological Chemistry (2015)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: P1B-ATPases; copper transport; gene expression; membrane transport; metal homeostasis
Affiliations: From the Institute of Experimental Biology, Department of Plant Molecular Physiology, and ., From the Institute of Experimental Biology, Department of Plant Molecular Physiology, and., Institute of Experimental Biology, Department of Genetics and Cell Physiology.

Abstract:  Plant copper P1B-type ATPases appear to be crucial for maintaining copper homeostasis within plant cells, but until now they have been studied mostly in model plant systems. Here, we present the molecular and biochemical characterization of two cucumber copper ATPases, CsHMA5.1 and CsHMA5.2, indicating a different function for HMA5-like proteins in different plants. When expressed in yeast, CsHMA5.1 and CsHMA5.2 localize to the vacuolar membrane and are activated by monovalent copper or silver ions and cysteine, showing different affinities to Cu(+) (Km ∼1 or 0.5 μM, respectively) and similar affinity to Ag(+) (Km ∼2.5 μM). Both proteins restore the growth of yeast mutants sensitive to copper excess and silver through intracellular copper sequestration, indicating that they contribute to copper and silver detoxification. Immunoblotting with specific antibodies revealed the presence of CsHMA5.1 and CsHMA5.2 in the tonoplast of cucumber cells. Interestingly, the root-specific CsHMA5.1 was not affected by copper stress, whereas the widely expressed CsHMA5.2 was up-regulated or down-regulated in roots upon copper excess or deficiency, respectively. The copper-induced increase in tonoplast CsHMA5.2 is consistent with the increased activity of ATP-dependent copper transport into tonoplast vesicles isolated from roots of plants grown under copper excess. These data identify CsHMA5.1 and CsHMA5.2 as high affinity Cu(+) transporters and suggest that CsHMA5.2 is responsible for the increased sequestration of copper in vacuoles of cucumber root cells under copper excess. 

Retracted: Yes

PubPeer comments:
 Posting a concern raised by another reader about Figure 7. The OVA + PBS panel and the OVA + imDC panel appear to show an area of overlap. Marked with red boxes. ![file] , 
 In addition, I noticed that in Figure 1C, the D0 panels of  With stroma and  Without stroma appear to look more similar than expected. Marked in blue. Image made lighter to bring out more details. The D0 and D6 panels of  Without stroma also look very similar, but these are cells in monolayer (not in solution) that were dying, so this is expected and not of concern. ![file] , 
 #1 I could not find an explanation in the text or the Figure Legend of the insets within three panels of Fig 7. I hope they are not intended to be restained enlargements of the smaller rectangles central to the panels, as these insets are the same for the OVA + PBSand OVA + imDC panels (apart from a 180-deg rotation). ![file] Not adding anything to Comment #1, but here is my attempt to dramatise the overlap between the two panels. ![file] , 
 This article has been corrected. See: https: onlinelibrary.wiley.com doi full 10.1002 eji.202070077 The correction states:  *In the aboveu2010mentioned article the D0, D6 and D12 fluorescent images of the u201cwithout stromau201d group in Figure 1C were duplicates of the D0 u201cwith stromau201d data. In addition, in Figure 7C, the brightfield hematoxylin and eosin (H E) images of lung sections of the u201cOVA+imDCu201d group was a duplication of the image of the u201cOVA+PBSu201d group. The authors have provided the original source data for Figure 1C and the corrected Figure 1 is provided below. For Figure 7C, the authors were unable to locate the original data for the experiment. As such, following a request by the editorial team, the authors have repeated the experiments presented in Figure 7C and have provided the raw data and an additional quantification for this figure. Quantification analysis was not part of the original publication; however, the Editors felt that such an analysis was essential in order to validate the choice of representative images. The corrected Figure 1 and Figure 7 are shown below along with the methods for the analysis. The raw data and analysis are included as Supporting Information. The repeated experiments in Figure 7 validate the conclusions of the paper. * ,