TOR1 and TOR2 have distinct locations in live cells

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The Target of Rapamycin (TOR) kinase is a crucial regulator of cell growth, existing in two distinct complexes.
• Previous studies on TOR localization in live cells were limited, leading to ambiguity regarding its precise cellular positioning.
• Understanding TOR localization is key to deciphering its role in diverse cellular processes.

Purpose of the Study:

• To investigate the endogenous localization of TOR1 and TOR2 in live Saccharomyces cerevisiae cells.
• To determine if TOR proteins retain function after internal tagging with green fluorescent protein (GFP).
• To correlate distinct localization patterns with the specific functions of TOR complexes.

Main Methods:

• Internal tagging of endogenous TOR1 and TOR2 genes with a 3XGFP cassette in S. cerevisiae.
• Functional validation of tagged TOR1 and TOR2 proteins through phenotypic assays (rapamycin sensitivity, cold sensitivity, manganese toxicity).
• Live-cell imaging to visualize the subcellular localization patterns of TOR1-3XGFP and TOR2-3XGFP.

Main Results:

• Internal tagging of TOR1 and TOR2 with 3XGFP did not impair their function, confirmed by multiple assays.
• TOR1-3XGFP exhibited diffuse cytoplasmic localization with concentration near the vacuolar membrane.
• TOR2-3XGFP showed cytoplasmic localization with predominant punctate signals at the plasma membrane.

Conclusions:

• TOR1 and TOR2 proteins possess distinct subcellular localization patterns in live yeast cells.
• The observed distinct localizations support the hypothesis that TOR1 and TOR2 function within separate complexes to regulate different cellular processes.
• This study provides critical insights into the spatial organization of TOR signaling pathways, essential for cell growth regulation.