Post-transcriptional and Post-translational Mechanisms of Autophagy Regulation

Macroautophagy (hereafter, autophagy) is a conserved catabolic process of cellular recycling essential for maintaining metabolic homeostasis. Autophagy allows for the selective or non-selective sequestration of cytoplasmic components in phagophores that mature to form double-membrane autophagosomes. This is followed by the delivery of the cargo-containing autophagosome to the degradative organelle for cargo breakdown. Selective autophagy plays a crucial role in removing damaged/superfluous cellular components. Non-selective autophagy targets random segments of the cytoplasm to the degradative organelle primarily in response to nutrient deficiency. During acute starvation, cells lack a supply of building blocks for synthesizing essential macromolecules. To prevent a collapse of cellular function, autophagy is upregulated in response to starvation via metabolic signals that integrate nutritional cues. This allows for the degradation of pre-existing macromolecules such as proteins, from the cytoplasmic portions delivered by autophagy, and the subsequent release of simple metabolites such as amino acids as breakdown products. These are then transported out of the organelle and into the cytoplasm by dedicated transporter proteins, allowing for new macromolecular synthesis. This makes autophagy a critical pathway in helping cells combat starvation. These roles of autophagy, its interaction with metabolism, where both influence each other, and the regulatory processes involved have been discussed in Chapter 1. A detailed description of the mechanisms of selective autophagy has been discussed in Chapter 4. The autophagy pathway involves multiple steps that are completed by the concerted action of numerous proteins. Therefore, simpler systems such as the baker’s yeast Saccharomyces cerevisiae are critical to understanding this pathway, especially because most components and mechanism of the machinery are conserved. Even in yeast, over 40 proteins are involved in autophagy highlighting the complexity of the pathway. Additionally, the function of these proteins must be exquisitely regulated to ensure timely autophagy induction and execution while preventing excess self-degradation which could be lethal. In Chapters 2 and 3 I discuss two regulatory mechanisms in yeast. In Chapter 2, I discuss how autophagy is regulated in yeast in response to two distinct nutritional challenges: nitrogen starvation and amino acid starvation. I find that autophagy is more highly upregulated during nitrogen starvation relative to amino acid starvation and that this regulation occurs at the post-transcriptional level. I focus on the protein kinase – Atg1 – involved in autophagy induction and find that nitrogen starvation differentially promotes Atg1 expression whereas ATG1 transcription remains comparable between the two conditions. I then explore the mechanism of post-transcriptional upregulation of Atg1 during nitrogen starvation and find that the kinase Rad53 and the RNA-binding protein Ded1 are responsible for promoting facile Atg1 production during nitrogen starvation. Finally, I show that ULK1, a mammalian homolog of Atg1, is similarly post-transcriptionally regulated by DDX3, the mammalian homolog of Ded1 – highlighting the conservation of this mechanism. A second component of the autophagy pathway – the lipid kinase Vps34 – is my focus for Chapter 3. I show that while Vps34 activity is essential for autophagy, hyperactivation of Vps34 reduces autophagy flux. I confirm that this effect is not due to transcriptional regulation because ATG gene expression is not affected by Vps34 hyperactivation. Indeed, I find that Vps34 blocks the fusion of the autophagosome with the vacuole. In summary, this thesis portrays mechanisms of autophagy regulation in yeast and provides clues regarding how differential regulation occurs during distinct nutritional challenges.

Topic: Autophagy in Disease and Therapy
Subfield: Epidemiology
Field: Medicine
Domain: Health Sciences