TOR Complex 1 (TORC1) is required for growth, while TORC2 regulates actin cytoskeletal polarization. The regulation and function of TORC1 is well understood, but TORC2 regulation has not been fully characterized; nor, is it clear if TORC2 is required for all actin-mediated events. We examined the role of TORC2 during the actin-dependent process of nutrient capture by phagocytosis in Dictyostelium and tested if TORC2 were involved.We show that loss of TORC2 components Rictor/Pia, SIN1/RIP3, and LST8 promotes phagocytosis, while inactivation of TORC1, by depletion of TORC1-specific Raptor, has no effect on phagocytosis. We also show that rates of phagocytosis are uncoupled from in vivo TORC1 activity. Also, while rapamycin is suggested to be a specific inhibitor of TORC1, our additional data indicate that rapamycin is also able to inhibit TORC2 regulation of phagocytosis, albeit indirectly. Thus, although TORC2 is required for cell polarization, it is not required for all actin-dependent processes. Finally, we suggest that the balanced regulations of TORC2 and TORC1 may be critical to coordinate and optimize growth with energy needs.? We have collaborated with Dr. C. Londos, LCDB/NIDDK, to study the function of proteins that associate specifically with the intracellular lipid storage droplets (LSDs) that assemble triacylglycerols and cholesteryl esters for energy metabolism, steroid hormone synthesis, membrane biosynthesis, and cell signaling. We had identified the novel PAT domain as a defining feature for LSD proteins Perilipin (Peri), ADRP, TIP47, discovered new family members (PAT5), and determined the first structure of a PAT family member, TIP47. The structural relationships among these various proteins had necessitated a new nomenclature. Accordingly we have proposed the following: Perilipin is now Perilipin 1, ADRP is Perilipin 2, TIP47 is Perilipin 3, S3-12 is Perilipin 4, and PAT5 is Perilipin 5. Functional studies using native and mutated forms of Peri1 confirmed its role in PKA-mediated lipolysis of triacylglycerols and showed that Peri is essential for the translocation of hormone-sensitive lipase (HSL) during lipolytic activation, but that while the adipose mass of peri-null mice is <30% that of WT, the mutant mice are susceptible to peripheral insulin resistance in liver, but not muscle. We also show that Peri3 (TIP47) is unable to functionally compensate for Peri1 but can complement the role of Peri2 (ADRP) in a cell culture model system. To analyze the various functions of the 5 genes that comprise the Peri family of mammalian proteins, we initiated a systematic series for production of singly and multiply targeted mutations of these genes in mouse ES cells and for their germline transmission in mice. To this end we now have Peri1(-/-) mice, and mutations in ES cells and mice for Peri2, 3, and 5. Others data had asserted that Peri2 (-/-) mice only had a limited phenotype, but an aberrant Peri2 product produced in those mice may have had a compensating effect. We now show by co-immunoprecipitation experiments a link of Peri2 to a pathway that is independent of Peri3 and suggest a more complex and non-overlapping regulatory mode for these highly related proteins.
