Vac8 Controls Vacuolar Membrane Dynamics during Different Autophagy Pathways in Saccharomyces cerevisiae

Abstract

The yeast vacuole is a vital organelle, which is required for the degradation of aberrant intracellular or extracellular substrates and the recycling of the resulting nutrients as newly available building blocks for the cellular metabolism. Like the plant vacuole or the mammalian lysosome, the yeast vacuole is the destination of biosynthetic trafficking pathways that transport the vacuolar enzymes required for its functions. Moreover, substrates destined for degradation, like extracellular endocytosed cargoes that are transported by endosomes/multivesicular bodies as well as intracellular substrates that are transported via different forms of autophagosomes, have the vacuole as destination. We found that non-selective bulk autophagy of cytosolic proteins as well as the selective autophagic degradation of peroxisomes (pexophagy) and ribosomes (ribophagy) was dependent on the armadillo repeat protein Vac8 in Saccharomyces cerevisiae. Moreover, we showed that pexophagy and ribophagy depended on the palmitoylation of Vac8. In contrast, we described that Vac8 was not involved in the acidification of the vacuole nor in the targeting and maturation of certain biosynthetic cargoes, like the aspartyl-protease Pep4 (PrA) and the carboxy-peptidase Y (CPY), indicating a role of Vac8 in the uptake of selected cargoes. In addition, we found that the hallmark phenotype of the vac8 strain, namely the characteristic appearance of fragmented and clustered vacuoles, depended on the growth conditions. This fusion defect observed in standard glucose medium can be complemented by the replacement with oleic acid or glycerol medium. This complementation of vacuolar morphology also partially restores the degradation of peroxisomes. In summary, we found that Vac8 controlled vacuolar morphology and activity in a context- and cargo-dependent manner.

Lifespan Increase of Podospora anserina by Oleic Acid Is Linked to Alterations in Energy Metabolism, Membrane Trafficking and Autophagy

“…Since in fungi the vacuole is the common compartment in which the different autophagic cargo is degraded, and since vacuoles were found to be affected in the PaAtg24 deletion mutant [9], we surmised that the common process impaired in ∆PaAtg24 is linked to the formation of vacuoles. This idea is supported by a recent study with yeast, in which oleic acid abrogates fragmented vacuole morphology and fusion defects in the vacuolar ∆Vac8 mutant [36].…”

Autophagy Stimulus-Dependent Role of the Small GTPase Ras2 in Peroxisome Degradation

“…The generation and induction of the plasmids Pex11-GFP [ 17 ], Ras2(G19V)/Ras2 ON , and Ras2(G22A)/Ras2 OFF [ 15 ] have been described in the corresponding publications.…”

“…The analysis of live cells was performed with a Zeiss Axioplan microscope and deconvolved with AxioVision 4.1 software (Zeiss, Jena, Germany) [ 5 , 17 ].…”

“…To analyze pexophagy [ 17 , 19 , 20 ], yeast strains expressing the peroxisomal membrane protein Pex11 C-terminally fused with GFP were grown in two precultures (20 mL overnight and 50 mL for 8 h, OD 600 nm = 0.3) in SD-medium at 30 °C. Peroxisomal proliferation was induced by incubating the cells for 16 h at 30 °C (OD 600 nm = 0.5) in 100 mL oleate media.…”

Nucleophagy—Implications for Microautophagy and Health

“…The competitive interaction with Atg13 and Nvj1 differentially regulates the Cvt and PMN pathways [ 66 , 67 ]. Vac8 is required for many micro- and macroautophagic modes [ 68 , 69 , 70 , 71 , 72 ]. The unique composition of the NVJ is at least in part due to Nvj1, which recruits various other proteins.…”