Atg8 regulates vacuolar membrane dynamics in a lipidation-independent manner in Pichia pastoris
Results Deletion of ATG8 results in abnormal vacuolar shape P. pastoris can use methanol as the sole carbon and energy source. Adaptation of glucose-grown P. pastoris cells to methanol medium leads to a great increase in the number and size of peroxisomes (van der Klei et al., 2006) . In this study, we observed a morphological change in the vacuole during the methanol adaptation period. A wild-type strain shifted to methanol medium for 2 hours contained a single spherical vacuole per cell,
... uole per cell, compared with 2-4 clustered vacuoles per cell when growing on glucose medium (Fig. 1A) . To examine the morphological change after methanol adaptation of P. pastoris in greater detail, we performed a morphometric analysis during the transition process by counting the cells exhibiting a single spherical vacuole over time (Fig. 1B) . FM4-64labeled P. pastoris cells under methanol adaptation were sampled, and fluorescence images were immediately taken. It was found that the number of cells containing a single round vacuole increased immediately following the shift to methanol medium, and the Summary Atg8 is a ubiquitin-like protein that is required, along with its lipidation system, for autophagy in all eukaryotic cells. The lipidated form of Atg8 is anchored on the autophagosomal membrane during autophagy. Here, we demonstrate a previously unknown role for Atg8 in vacuolar membrane dynamics. In the methylotrophic yeast Pichia pastoris, vacuoles were found to fuse to become a single spherical vacuole during adaptation from glucose-to methanol-containing medium. Atg8 is responsible for the vacuolar fusion in P. pastoris during this adaptation to methanol. Although vacuole fusion required processing of Atg8 at the C-terminus, it did not require lipidation of Atg8 for autophagy. This is the first report of the function of any Atg8 protein family member in a process other than autophagy that is independent of lipidation.