Fig. 7
From: Genetic dissection of the mitochondrial lipoylation pathway in yeast
Growth assay and western blot analysis of Lip3 transfer activity with truncated Lat1 as the lipoyl donor. All plasmids transformed into the different strains are based on the YEp352 yeast episomal plasmid backbone. a Growth assay of wild-type+YEp352, Δgcv3Δlat1 + YEp352-LplAK133L, Δgcv3Δlat1 + YEp352-LplA, Δgcv3Δlat1 + N-Lat1 + YEp352-LplAK133L, and Δgcv3Δlat1 + N-Lat1 + YEp352-LplA. Cells were grown for 16 h, adjusted to an OD600 of 0.5 and spotted as 1× 1/10x, 1/100×, and 1/1000× serial dilutions SCD-URA as the general growth control, as well as on SCG-URA, SCG-URA + 100 μM LA, or SCG-URA + 100 μM C8. Plates were incubated for 4 days at 30 °C. For western blot analysis, whole cell extracts were collected from cells grown in YPG with and without 100 μM LA or C8 supplementation. b Whole cell extracts of wild-type+YEp352, Δgcv3Δlat1 + LplA, and Δgcv3Δlat1 + LplAK133L strains were analyzed with anti-LA serum and anti-actin serum for the loading control. Please note that the white line separating lane 4 and 5 in the lipoylation signal data is not a splicing artifact, but an artifact of the overlay with the image file containing the size marker information. All samples are from the same western blot and are continuous (see Additional File Fig. A4), and the identical membrane was used to probe for the actin loading control. c Whole cell extracts of the Δgcv3Δlat1 strain expressing the His6- and C-protein double-tagged N-Lat1 lipoyl domain plus either the YEp352-LplA or YEp352-LplAK133L plasmids were analyzed on immunoblots. The anti-His6 signal indicates the presence of the His-tagged N-Lat1 lipoyl domain protein fragment. The blots were reacted with anti-LA serum, anti-His serum, and anti-actin serum for the loading control