Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which grow in size and further expand to form autophagosomes, engulfing cellular material to be degraded by fusion with lysosomes (vacuole in yeast). Autophagosomal membranes do not bud from a single cell organelle, but are generated de novo. Several lipid sources for autophagosomal membranes have been identified, but the whole process of their generation is complex and not entirely understood. In this study, we investigated how the mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), affects autophagy in yeast. We show that POR1 deficiency reduces the autophagic capacity and leads to changes in vacuole and lipid homeostasis. We further investigated whether limited phosphatidylethanolamine (PE) availability in por1∆ was causative for reduced autophagy by overexpression of the PE-generating phosphatidylserine decarboxylase 1 (Psd1). Altogether, our results show that POR1 deficiency is associated with reduced autophagy, which can be circumvented by additional PSD1 overexpression. This suggests a role for Por1 in Psd1-mediated autophagy regulation.

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The dynamic nature of the actin cytoskeleton controls many cellular processes and a loss of its plasticity has been linked to accelerated cell ageing. Cofilin is an actin-binding protein that controls actin dynamics and is linked to mitochondrial signalling pathways that control drug resistance and cell death. Here we show that cofilin driven stabilisation of actin dynamics leads to a loss in cell wall integrity, vacuole fragmentation and disruption of lipid homeostasis, lipid droplet accumulation and the promotion of necrosis. Our data suggest that these phenotypes are triggered by the inappropriate activation of Protein Kinase C (PKC) which leads to constitutive MAPK signalling. Cofilin-driven activation of the MAPK Slt2 requires the presence of the voltage dependent anion channel (VDAC), which resides in the mitochondrial outer membrane, referred to as Porin 1 (Por1) in yeast. This provides further evidence for a link between actin regulation and mitochondrial signalling. Porin has recently been implicated in lipid transport and we find that prevention of excessive lipid droplet accumulation, achieved by deleting the LRO1 and DGA1 genes, was sufficient to prevent Slt2 activation and restore cellular homeostasis in actin stabilised cells. These data suggest that the integrity of the actin cytoskeleton is essential to maintain the fidelity of MAPK signalling and that this in turn is crucial for the maintenance of lipid homeostasis and cell health in S. cerevisiae. Our data also suggest that chronic actin stabilisation leads to mitochondrial VDAC-dependent Slt2 activation and altered, pro-death, cell fate in yeast cells.

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