RESUMO
The lysosome (or vacuole in fungi and plants) is an essential organelle for nutrient sensing and cellular homeostasis. In response to environmental stresses such as starvation, the yeast vacuole can adjust its membrane composition by selectively internalizing membrane proteins into the lumen for degradation. Regarding the selective internalization mechanism, two competing models have been proposed. One model suggests that the ESCRT machinery is responsible for the sorting. In contrast, the ESCRT-independent intralumenal fragment (ILF) pathway proposes that the fragment generated by homotypic vacuole fusion is responsible for the sorting. Here, we applied a microfluidics-based imaging method to capture the complete degradation process in vivo. Combining live-cell imaging with a synchronized ubiquitination system, we demonstrated that ILF cargoes are not degraded through intralumenal fragments. Instead, ESCRTs function on the vacuole membrane to sort them into the lumen for degradation. We further discussed challenges in reconstituting vacuole membrane protein degradation.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Ferritinas/genética , Ferritinas/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Lisossomos/genética , Proteínas de Membrana/genética , Técnicas Analíticas Microfluídicas , Microscopia de Fluorescência , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Tempo , Imagem com Lapso de Tempo , Ubiquitinação , Vacúolos/genéticaRESUMO
Cellular adaptation in response to nutrient limitation requires the induction of autophagy and lysosome biogenesis for the efficient recycling of macromolecules. Here, we discovered that starvation and TORC1 inactivation not only lead to the up-regulation of autophagy and vacuole proteins involved in recycling but also result in the down-regulation of many vacuole membrane proteins to supply amino acids as part of a vacuole remodeling process. Down-regulation of vacuole membrane proteins is initiated by ubiquitination, which is accomplished by the coordination of multiple E3 ubiquitin ligases, including Rsp5, the Dsc complex, and a newly characterized E3 ligase, Pib1. The Dsc complex is negatively regulated by TORC1 through the Rim15-Ume6 signaling cascade. After ubiquitination, vacuole membrane proteins are sorted into the lumen for degradation by ESCRT-dependent microautophagy. Thus, our study uncovered a complex relationship between TORC1 inactivation and vacuole biogenesis.
