Figure 4. PRMT1 inhibits the GAP activity of GATOR1.

A, Structure of the GATOR1 complex (Protein Data Bank code 6CET) displayed by PyMOL.

B-C, PRMT1 methylates NPRL2 and antagonizes the GAP activity of the GATOR1 complex. Purified GATOR1 complex was subjected to in vitro methylation assays with PRMT1 WT or mutants (G98R, E166Q), with or without PRMT1 inhibitors, and analyzed via the MTase-Glo^™ Methyltransferase Assay kit (B) or immunoblotting with the specific antibody for NPRL2 R78me2a (C). n=3 biological repeats, ***p<0.001, unpaired, two-tailed Student’s t-test.

D, PRMT1-mediated asymmetric di-methylation of NPRL2 impairs the GAP activity of the GATOR1 complex. The purified GATOR1 complex was subjected to in vitro methylation assays as in f and was further subjected to GAP activity analysis. n=3 biological repeats, ***p<0.001, unpaired, two-tailed Student’s t-test.

E, The activation of mTORC1 by methionine is decreased in cells expressing the NPRL2 K75A and L82A mutants. NPRL2 null HEK293T cells were infected with either wild-type NPRL2 or the indicated mutants, starved of methionine for 2 hours, restimulated with methionine (100 μM) for 20 min, and analyzed via immunoblotting with the indicated antibodies.

F, Coomassie blue staining of the purified GATOR1 complex containing either wild-type NPRL2 or the indicated mutants.

G, Mutation of the flanking residues near R78 in NPRL2 protein attenuated PRMT1-mediated methylation. The purified GATOR1 complex containing either wild-type NPRL2 or the indicated mutants were subjected to in vitro methylation assays as described in B. n=3 biological repeats, ***p<0.001, unpaired, two-tailed Student’s t-test.

H, Methylation of R78 in NPRL2 protein is essential for PRMT1-mediated inhibition of the GAP activity of GATOR1. The purified GATOR1 complex containing either wild-type NPRL2 or the indicated mutants were subjected to in vitro methylation assays as described in B. n=3 biological repeats, ***p<0.001,**p<0.01, unpaired, two-tailed Student’s t-test.

I-J, SAMTOR blocked PRMT1-mediated inhibitory effect on GATOR1 activity in vitro. The purified GATOR1 complex was subjected to in vitro methylation assays as indicated and was analyzed via the MTase-Glo^™ Methyltransferase Assay kit (I) or immunoblotting with the specific antibody for NPRL2 R78me2a (J). n=3 biological repeats, ns, no significant difference, ***p<0.001, **p<0.01, unpaired, two-tailed Student’s t-test.

K, SAMTOR D190A mutant, but not wild-type, blunted PRMT1-mediated NPRL2 asymmetric di-methylation in cells.

L, A schematic model depicting PRMT1-mediated NPRL2 asymmetric di-methylation in methionine sensing of mTORC1 signaling.

See also Figure S4.