Fig 2: A phospho-mimetic mutation of Sx16-T7 exhibits reduced interaction with mVps45.Identical amounts of recombinant Protein A-tagged Sx16 wild type or Sx16-T7D were used to pull down HA-tagged mVps45 over-expressed in yeast. A constant amount (250 µg) of yeast lysate expressing HA-mVps45 was incubated with decreasing amounts (20, 15, 10, 5, 2.5, and 1 µg) of either Sx16-PrA or Sx16-T7D-PrA for 2 h on ice. PrA-tagged species were then captured using IgG-Sepharose beads, washed and eluted by boiling in SDS-PAGE sample buffer. Samples were then stained for total protein (A) or immunoblotted using anti-HA (B). In the figure, L refers to starting Lysate and B shows a sample of IgG beads ( 10x more than used in the capture lanes) to control for any non-specific binding. PrA alone did not exhibit any binding to HA-mVps45 (uncropped blots can be found in the Supplemental Information). Data from a representative binding experiment is shown, together with quantification of three separate experiments of this type presented in (C) as mean ± S.D. Note that in the quantification, the data are normalised relative to the level of mVps45 binding observed using 20 µg of Sx16. The approximate positions of molecular weight markers are indicated (in kDa).

Fig 3: Sx16-T7D reduces insulin-stimulated glucose transport.3T3-L1 adipocytes were infected with lentivirus delivering either wild-type Sx16, or Sx16-T7A or Sx16-T7D as outlined in Methods. (A) DeGlc uptake was assayed after incubation with or without 1 µM insulin for 30 min. Shown are the means of three independent experiments in which basal and insulin-stimulated deGlc uptake rates were measured in quadruplicate at each condition and are presented as a % of the insulin-stimulated rate in control (non-infected) cells. Over-expression of Sx16-T7D consistently impaired insulin-stimulated deGlc uptake; p = 0.02 compared to control cells (ANOVA). No other differences were observed between groups. (B) Shown are lysates from a typical dataset immunoblotted with anti-Sx16, anti-GLUT4, anti-mVps45 and anti-GAPDH. Note that over-expressed Sx16 and mutants thereof consistently migrate faster than the endogenous protein (indicated by * on the figure), the approximate positions of molecular weight markers are indicated (in kDa). We saw no significant differences in levels of expression of the different Sx16 mutants across all experiments of this type. (C) 3T3-L1 adipocytes expressing either Sx16-WT, T7A or T7D (as indicated) were treated with or without 1 µM insulin for 30 min and lysates separated on SDS-PAGE and immunoblotted using antibodies that recognise phosphorylated Akt or total Akt, as shown. Data from a typical experiment is shown.