Nedd4, a human ubiquitin ligase, affects actin cytoskeleton in yeast cells

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Human Nedd4 ubiquitin ligase plays roles in protein trafficking, signal transduction, and oncogenesis.
• Nedd4 with an inactive WW4 domain exhibits toxicity in yeast cells.
• The actin cytoskeleton is crucial for various cellular processes, including cell shape, motility, and division.

Purpose of the Study:

• To investigate the effects of human Nedd4 ubiquitin ligase expression on the yeast actin cytoskeleton.
• To determine the role of Nedd4's catalytic and WW4 domains in its cellular toxicity and impact on actin.
• To explore the interaction between Nedd4 and the actin regulatory protein Las17.

Main Methods:

• Yeast genetics and cell biology techniques were employed.
• Phenotypic analysis of yeast cells expressing Nedd4 and Nedd4w4 variants.
• Sensitivity assays using Latrunculin A, an actin-depolymerizing drug.
• Western blot analysis to determine protein levels, including Las17p.

Main Results:

• Expression of NEDD4 or NEDD4w4 genes caused abnormal actin cytoskeleton and increased sensitivity to Latrunculin A in yeast.
• These phenotypes were less pronounced when the catalytic domain of Nedd4 was inactivated.
• Overexpression of LAS17 was detrimental to NEDD4w4-expressing cells, and Nedd4w4 increased Las17p levels, partly dependent on its catalytic domain.
• Nedd4 variants and LAS17 overexpression suppressed the growth defect of the arp2-1 strain.

Conclusions:

• Human Nedd4 ligase inhibits yeast cell growth by disturbing the actin cytoskeleton, partly through increasing Las17p levels.
• Nedd4's ubiquitination targets likely include evolutionarily conserved actin cytoskeleton-associated proteins.
• The catalytic activity of Nedd4 is important for its effects on the actin cytoskeleton and cell growth.