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. 2015 Oct 6;290(49):29506–29518. doi: 10.1074/jbc.M115.684233

FIGURE 2.

FIGURE 2.

Identification of Atg homologs and probes for monitoring autophagy in K. marxianus. A, Atg homologs in K. marxianus. Most KmAtg proteins are shorter than their ScAtg counterparts (red). Asterisk, mature form of Atg8. B, KmAtg8 is required for autophagosome formation in K. marxianus. K. marxianus wild-type (WT), Kmpep4Δ, and Kmpep4Δ Kmatg8Δ cells were grown at 30 °C, treated with rapamycin for 4 h, and subjected to electron microscopy. C, KmAtg8 is essential for K. marxianus autophagy. K. marxianus wild-type (WT), Kmpep4Δ Kmprb1Δ, and Kmatg8Δ cells expressing an N-terminally truncated variant of KmPho8 (KmPho8ΔN44) were grown at 30 °C and then shifted to nitrogen-starvation medium. Kmpho8Δ cells were used as a control not expressing the probe KmPho8ΔN44 (without KmPho8ΔN44). After starvation for 5 h, the cells were harvested, and ALP activities were measured. a.u., arbitrary unit. D, GFP-KmAtg8 is transported into the vacuole. Wild-type K. marxianus cells expressing GFP-KmAtg8 (green) were treated with FM4-64 (magenta). The cells were grown at 30 °C (Nut.) and then treated with rapamycin for 2 h (Rap. 2 h) or incubated in nitrogen-starvation medium for 2 h (Stv. 2 h). The cells were observed by fluorescence microscopy. E, C-terminal regions of ScAtg8 and KmAtg8. Atg8 is synthesized as a precursor form with an additional segment at its C terminus (blue) and cleaved by Atg4 to be converted into the mature form. KmAtg8FG is a truncated variant that mimics a mature form. KmAtg8FA contains an Ala substitution at residue 116 (red). F, KmAtg4 is responsible for the maturation of KmAtg8. GFP-KmAtg8, GFP-KmAtg8FG, and GFP-KmAtg8FA (green) were expressed in K. marxianus wild-type and Kmatg4Δ cells. The cells were treated with FM4-64 (magenta), treated with rapamycin for 2 h, and then observed by fluorescence microscopy. G, GFP-processing assay in K. marxianus. The K. marxianus cells used in F were treated with rapamycin for 1 h, and then total lysates were prepared. Samples were subjected to immunoblot analysis using anti-GFP antibody and streptavidin-HRP as a loading control (biotinylated). proc.GFP indicates a processed form of the GFP moiety.