A putative

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Nuclear envelope morphology protein 1 (NEM1) and phosphatidate phosphatase (PAH1) are key regulators of lipid homeostasis and membrane biogenesis in model organisms.
• Understanding the roles of these proteins in diverse eukaryotes like Tetrahymena thermophila is crucial for a comprehensive view of cellular regulation.

Purpose of the Study:

• To investigate the functions of four putative NEM1 homologues (TtNEM1A, TtNEM1B, TtNEM1C, TtNEM1D) in the Tetrahymena thermophila genome.
• To determine the relationship between Tetrahymena NEM1 homologues and PAH1 in regulating cellular processes.
• To compare the functions of Tetrahymena NEM1 homologues with their counterparts in yeast and mammals.

Main Methods:

• Gene knockout studies to assess the essentiality and function of TtNEM1 homologues.
• Overexpression of Tetrahymena PAH1 to test for functional rescue of Nem1 loss-of-function phenotypes.
• Microscopy to evaluate the impact on ER/nuclear morphology and lipid droplet number.
• Functional complementation assays by expressing Tetrahymena NEM1 homologues in yeast.

Main Results:

• TtNEM1A is essential for Tetrahymena growth, as knockout strains could not be generated.
• Disruption of TtNEM1B, but not TtNEM1C or TtNEM1D, led to a reduced number of lipid droplets.
• PAH1 overexpression rescued the loss of TtNem1B function, indicating a conserved regulatory pathway.
• Unlike in other organisms, Tetrahymena NEM1 homologues did not regulate ER/nuclear morphology.
• Tetrahymena NEM1 homologues could not functionally replace yeast NEM1.

Conclusions:

• A conserved cascade involving NEM1 and PAH1 regulates lipid homeostasis and membrane biogenesis in Tetrahymena.
• Tetrahymena possesses a unique Nem1-independent function of Pah1 in regulating ER morphology.
• Despite conserved roles in lipid homeostasis, Tetrahymena NEM1 homologues exhibit distinct functional specificities compared to yeast and mammals.