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. 2019 Aug 27;9:12442. doi: 10.1038/s41598-019-48866-y

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Temperature and copy number dependent complementation by HHD-mutant hSPCA1a. Yeast strain PAP4920 (pmc1Δ pmr1Δ cnb1Δ) expressing no hSPCA1a (empty vector, EV), wt hSPCA1a (WT) or the indicated hSPCA1a HHD variants from either the integrative plasmid pPAP5480 (copy number of one, rows 1 and 2 in each picture) or the replicative plasmid pPAP4997 (copy number of 20, rows 3 and 4 in each picture) as indicated in the top pane, were spotted on agar-plates containing the media shown on the left of the lower pane. All media are synthetic defined media containing either glucose or galactose as the only carbon source as indicated. Media containing BAPTA (including 0 mM BAPTA) did not have any added CaCl₂. Plates were incubated at 15 °C, 20 °C, 25 °C, 30 °C or 35 °C as indicated, and photographed on a daily basis. Due to temperature dependent growth rates the selected pictures in panels Glucose +20 mM CaCl₂, Galactose +20 mM CaCl₂ and Galactose +0 mM BAPTA were taken at the earliest time points where yeast expressing wt ATP2C1a and yeast cells expressing no ATP2C1a had generated a clearly visible spot. The rest of the pictures were taken at time points where yeast cells expressing wt ATP2C1a had generated a visible spot and yeast cells expressing no ATP2C1a had failed to generate a spot. Results of one out of two experiments are shown.