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. Author manuscript; available in PMC: 2018 Dec 2.
Published in final edited form as: J Cell Sci. 2016 Sep 23;129(21):4067–4075. doi: 10.1242/jcs.195255

Figure 3. Ymr018w mediates targeting of a subset of PTS1 proteins to peroxisomes.

Figure 3

(A) Using an automated mating procedure we created an N’-tagged GFP-peroxisomal library with the deletion of YMR018W, PEX5 or PEX7 genes. Using a high content microscopy screen we examined the effect of the deletion on the localization of each of the peroxisomal proteins under growth on both glucose and oleate-containing media and could not detect an obvious effect of YMR018W deletion. Using the same automated mating procedure we created additional N’-GFP peroxisomal libraries with one of the following genomic modifications: mCherry-YMR018W overexpression (OE), Δpex5 + mCherry-YMR018W OE and Δpex7 + mCherry-YMR018W OE. A strain carrying a Neurseothricin selection was used as control for no modification in peroxisomal genes. We examined the effect of the different manipulations on the localization of each of the peroxisomal proteins and discovered three PTS1 proteins that were localized to peroxisomes when Ymr018w was over expressed either with a mCherry tag or when it was over expressed under a GPD promoter without a tag (no tag): (B) Mls1, (C) Mls2 and (D) Gto1. (E) A quantitative analysis of the percentage of cells (200 cells per strain) with GFP-Gto1 puncta in Δpex5, Δpex5+TEF2-mCherry-Ymr018w and Δpex5+GPDpr-Ymr018w strains, relative to a control strain with an intact PEX5 gene. Over expression of YMR018W increases the percentage of cells containing punctate Gto1 implying that Gto1 is a target of Ymr018w. (F) A strain expressing GFP with the last 10 amino acids of the Ymr018w-target Mls1 (MDH3pr-GFP-PTS1(Mls1)) was visualized. While GFP-PTS1(Mls1) appeared in puncta in the control strain and in the cytosol in the absence of PEX5, it resumed peroxisomal localization when Ymr018w was overexpressed. This indicates that the last 10 amino acids of Mls1 are sufficient to mediate targeting by Ymr018w. All images show a single focal plane.