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. 2022 Mar 17;18(3):e1010114. doi: 10.1371/journal.pgen.1010114

Fig 3. Cells devoid of TRAPPC10 display a membrane trafficking defect and lack detectable levels of TRAPPC9.

Fig 3

(A) Wild type HEK293 cells or TRAPPC10-/- cells either not transfected or transfected with FLAG-tagged wild type TRAPPC10 or one of the TRAPPC10 variants indicated were infected with VSVG-GFP ts045 4 hours after transfection. After an overnight incubation at 40°C, the cells were shifted to 32°C and imaged every minute. The fluorescence intensity in the Golgi was quantified and plotted versus time as described in the Materials and Methods section. The inset shows a western blot for the transfected proteins probed with anti-FLAG antibody. Representative images are shown in S7 Fig. (B) RFP-tagged versions of either wild type TRAPPC10, the p.Gly1131Valfs*19 variant or the p.Pro929Leu variant were transfected into HeLa cells and lysates prepared every day for 4 days. The lysates were fractionated by SDS-PAGE and probed with anti-mCherry antibody to reveal the RFP-tagged construct. Though the trends were consistent, statistical significance was seen only at day 4. The half-life for each overexpressed protein was found to be approximately 5.4 days, 4.4 days and 3.1 days for the wild type, p.Gly1131Valfs*19 and p.Pro929Leu variants, respectively. (C) RFP-tagged TRAPPC10 and the p.Gly1131Valfs*19 and p.Pro929Leu variants were transfected into HEK293 cells. The cells were either untreated (-) or treated (+) with 50 μm MG132 for 24 hours on the third day post-transfection. Lysates were then prepared, fractionated by SDS-PAGE and probed with anti-mCherry and tubulin as a loading control. (D) TRAPPC10-/- cells were either untransfected or transfected with FLAG-tagged wild type TRAPPC10, the p.Gly1131Valfs*19 variant or the p.Pro929Leu variant. After 24 hours, lysates were prepared and probed for the FLAG constructs to verify expression, TRAPPC9 and tubulin as a loading control. Parental HEK293 cells were also probed to assess the level of TRAPPC9 in the presence of TRAPPC10. (E) Lysates were prepared from lymphoblastoid cells from either control, an individual homozygous for the TRAPPC10 p.Gly1131Valfs*19 variant or three individuals homozygous for the TRAPPC9 p.(Arg475*) variant and subjected to western analysis to reveal the proteins indicated.