The entry of unclosed autophagosomes into vacuoles and its physiological relevance

It is widely stated in the literature that closed mature autophagosomes (APs) fuse with lysosomes/vacuoles during macroautophagy/autophagy. Previously, we showed that unclosed APs accumulated as clusters outside vacuoles in Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. However, the fate of such unclosed APs remains unclear. In this study, we used a combination of cellular and biochemical approaches to show that unclosed double-membrane APs entered vacuoles and formed unclosed single-membrane autophagic bodies after prolonged nitrogen starvation or rapamycin treatment. Vacuolar hydrolases, vacuolar transport chaperon (VTC) proteins, Ypt7, and Vam3 were all involved in the entry of unclosed double-membrane APs into vacuoles in Vps21-mutant cells. Overexpression of the vacuolar hydrolases, Pep4 or Prb1, or depletion of most VTC proteins promoted the entry of unclosed APs into vacuoles in Vps21-mutant cells, whereas depletion of Pep4 and/or Prb1 delayed the entry into vacuoles. In contrast to the complete infertility of diploid cells of typical autophagy mutants, diploid cells of Vps21 mutant progressed through meiosis to sporulation, benefiting from the entry of unclosed APs into vacuoles after prolonged nitrogen starvation. Overall, these data represent a new observation that unclosed double-membrane APs can enter vacuoles after prolonged autophagy induction, most likely as a survival strategy. Author summary Normal autophagy is critical for cellular homeostasis, whereas defective autophagy is closely linked to human disease development and progression. The model organism yeast has been greatly contributing to basic and applied researches of autophagy. Previously, we found that unclosed double-membrane APs accumulated as clusters outside vacuoles in haploid yeast Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. If these APs could not enter vacuoles to be degraded, the autophagy in these mutants would be completely interrupted to be defective. However, we found herein that the unclosed double-membrane APs in Vps21-mutant cells entered vacuoles to be degraded after prolonged autophagy induction or to be unclosed single-membrane autophagic bodies if further lacking vacuolar hydrolases. When we cultured diploid Vps21-mutant cells in a sporulation medium, their unclosed APs also entered vacuoles to be degraded, so that these diploid mutant cells could progress through meiosis to sporulation as diploid wild-type cells, not to be complete infertile as diploid cells of typical autophagy mutants. VTC proteins, Ypt7, and Vam3 were all important for the entry of unclosed double-membrane APs into vacuoles. Facilitating the entry of unclosed APs into lysosomes for degradation through autophagy may serve as a remedy strategy in autophagosome-closure-impaired diseases.

Automated summary

This study demonstrates that unclosed double-membrane autophagosomes can enter vacuoles after prolonged autophagy induction, potentially serving as a survival strategy. This finding provides a possible therapeutic strategy for diseases where autophagosome closure is impaired.