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A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Published on: July 18, 2011

Cancer-associated mutations in autophagy-related proteins analyzed in yeast and human cells

Yuchen Lei^1,2, Louise Uoselis^3,4, Dimitra Dialynaki¹

¹Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.

|February 28, 2025

Abstract:

Macroautophagy/autophagy is a conserved process among eukaryotes and is essential to maintain cell homeostasis; the dysregulation of autophagy has been linked with multiple human diseases, including cancer. However, not many studies have focused on the cancer-related mutations in ATG (autophagy related) proteins, which are likely to affect the protein function, influence autophagy activity and further contribute to the progression of the disease. In this study, we focused on the four ATG4 isoforms, which have a higher mutation frequency compared with the other core ATG proteins (i.e. those involved in autophagosome formation). We first studied the mutations in conserved residues and characterized one cancer-associated mutation that significantly impairs protein function and autophagy activity. Extending the study, we determined a region around the mutant residue to be essential for protein function, which had yet to be examined in previous studies. In addition, we created a yeast system expressing the human ATG4B protein to study mutations in the residues that are not conserved from human to yeast. Using this yeast model, we identified six cancer-associated mutations affecting autophagy. The effects of these mutations were further tested in mammalian cells using a quadruple ATG4 gene knockout cell line. Our study proves the principle of using human disease-associated mutations to study Atg proteins in yeast and generates a yeast tool that is helpful for a rapid screen of mutations to determine the autophagy phenotype, providing a new perspective in studying autophagy and its relation with cancer.Abbreviations: 4KO: ATG4 tetra knockout; ATG: autophagy related; BafA1: bafilomycin A₁; GFP: green fluorescent protein; LC3-II: PE-conjugated form of LC3B; ORF: open reading frame; PE: phosphatidylethanolamine; RFP: red fluorescent protein; SEP: superecliptic pHluorin; Ubl: ubiquitin-like; UCEC: uterine corpus endometrial carcinoma.

Insights

Cancer-associated mutations in autophagy-related (ATG) proteins disrupt cellular homeostasis. This study identifies specific ATG4 mutations impairing autophagy, using a novel yeast model to screen cancer-linked mutations affecting autophagy.

Area of Science:

Cell Biology
Molecular Biology
Genetics

Background:

Autophagy is a fundamental cellular process crucial for maintaining homeostasis, with its dysregulation implicated in human diseases like cancer.
While autophagy is vital, cancer-related mutations in autophagy-related (ATG) proteins, particularly ATG4 isoforms, are understudied despite their potential impact on protein function and disease progression.

Purpose of the Study:

To investigate the functional consequences of cancer-associated mutations in ATG4 proteins on autophagy activity.
To develop and utilize a yeast-based system for screening mutations in human ATG4B, especially non-conserved residues, and validate findings in mammalian cells.

Main Methods:

Characterization of mutations in conserved residues of ATG4 isoforms, including functional and autophagy activity assays.
Development of a yeast expression system for human ATG4B to screen for cancer-associated mutations affecting autophagy.
Validation of identified mutations in a quadruple ATG4 gene knockout (4KO) mammalian cell line.

Main Results:

A specific cancer-associated mutation in ATG4 was identified, significantly impairing protein function and autophagy.
A novel region essential for ATG4 protein function was determined.
The yeast model successfully identified six cancer-associated mutations affecting autophagy, which were subsequently validated in mammalian cells.

Conclusions:

The study validates the use of yeast models for studying human disease-associated mutations in ATG proteins.
A novel yeast tool was developed for rapid screening of mutations impacting autophagy, offering new insights into autophagy's role in cancer.

Read full analysis

Keywords:

ATG4 autophagy cancer mutation yeast analysis of human proteins

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