Extended Data Figure 7 (to Figure 3). Sec31a protects Sec13 from Skp1-mediated proteasomal degradation and supports T cell functional fitness.

(a) HA-tagged WT or the indicated lysine mutant constructs of Sec13 were transfected into HEK293T cells individually. Immunoblot analysis of HA and Hsp90. (b) HA-tagged WT or K260R mutant Sec13 was transfected into HEK293T cells together with the K48-only His-Ub followed by MG132 treatment and anti-HA immunoprecipitation (IP). Immunoblot analysis for HA, His-Ub and β-Actin. WCL, whole cell lysate. (c) Cas9-expressing CD4⁺ T cells were transduced with sgNTC or sgSec31a retrovirus (Ametrine⁺) together with WT or K260R mutant Sec13-expressing retrovirus (GFP⁺). Ametrine⁺GFP^lo cells (see gating on flow cytometry plot, upper) were stimulated with TCR for 0 or 3 h. Immunoblot analysis and quantification of relative Sec13 and p-S6 levels (n = 4 samples each group). (d) Volcano plot of proteins, including Skp1, that interact with HA-Sec13 in induced T_reg cells as identified by mass spectrometry (n = 3 samples each group). (e) Induced T_reg cells transduced with HA-tagged-Sec13- or empty vector-expressing retrovirus were lysed with CHAPS buffer followed by α-HA immunoprecipitation (IP) and immunoblot analysis of HA and Skp1. (f) Induced T_reg cells were lysed with CHAPS buffer followed by immunoprecipitation of endogenous Skp1 and immunoblot analysis of Skp1 and Sec13. (g) Interaction of endogenous Skp1 with Sec13 in sgNTC- or sgSec31a-transduced cells. (h) Naïve CD4⁺ T cells were stimulated with α-CD3/CD28 antibodies for 0, 24, 48 or 72 h. Immunoblot analysis of anti-Skp1 immunoprecipitants and WCL for Skp1, Sec13, and β-Actin (n = 2 samples per group). (i) Immunoblot analysis and quantification of relative expression of indicated proteins in sgNTC- or sgSkp1 (both Ametrine⁺)-transduced cells (n = 3 samples per group). (j) Indicated sgRNA-transduced cells were labelled with CellTrace Violet (CTV), and stimulated with α-CD3/CD28 antibodies for 72 h, followed by flow cytometry analysis and quantification of CTV dilution (n = 3 samples each group). (k) Diagram of SMARTA T cell transfer and LCMV infection system. Briefly, SMARTA–Cas9 CD4⁺ T cells (CD45.1⁺) transduced with sgRNA for candidate genes (CD45.1⁺Ametrine⁺) and mixed with sgNTC (CD45.1⁺mCherry⁺; ‘spike’)-transduced cells at a 1:1 ratio, and adoptively transferred into naïve (unchallenged; CD45.2⁺) mice that were left uninfected (see l) or challenged with LCMV infection (see Fig. 3f). (l) Quantification of the relative proportion (normalized to ‘spike’) of donor-derived (CD45.1⁺) T cells in the spleen of uninfected mice at 7 d after transfer (n =6 mice per group). (m) Cells transduced with sgNTC (Ametrine⁺), sgSec31a (Ametrine⁺) or sgSec31a-Skp1 (GFP⁺ and Ametrine⁺) were sorted and stimulated with α-CD3/CD28 antibodies for 20 h (n = 6-7 samples per group), followed by the measurement of extracellular acidification rate (ECAR). Oligo, oligomycin; FCCP, fluoro-carbonyl cyanide phenylhydrazone; Rot, rotenone. Mean ± s.e.m. (c, i, j, l, m). **P < 0.01; ***P < 0.001; one-way ANOVA (c, i, j, l, m, right); two-way ANOVA (m, left). Data are representative of one (d, j, l, m) or two (a, b, e–h), or pooled from two (c) or three (i) experiments.