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. 2021 Mar 22;40(8):e107238. doi: 10.15252/embj.2020107238

Figure 1. GOLPH3 interacts with the cytosolic tails of selected GSL synthetic enzymes.

Figure 1

  1. Pull‐down experiments using as a bait the biotinylated peptides mimicking the N‐terminal cytosolic tail of glycoenzymes performed from the whole HeLa cells lysates (top blot) or purified His‐tagged GOLPH3 protein (bottom blot). The N‐terminal cytosolic tail sequence of each glycoenzyme is indicated on the top of the blot (LxxR motif in red, biotin at the C‐terminus). Immunoblotting shows affinity‐captured GOLPH3 (endogenous and recombinant).
  2. Control or GOLPH3‐KD HeLa cells expressing luminal SI‐GFP or the chimeras of sucrose‐isomaltase containing the N‐terminal cytosolic tails of LCS, Gb3S, GM3S and GD3S were fixed and labelled for surface localised GFP (red). Scale bar, 20 μm.
  3. Pull‐down experiments of biotinylated LCS WT and LCS mutant cytosolic tails from HeLa cells lysates (top blot) or purified His‐tagged GOLPH3 protein (bottom blot). The N‐terminal cytosolic tail of LCS WT and LCS mutants (mut. 1, 2 and 3) is shown on the top of the blot (mutations are in blue, LxxR motif in red, biotin is at the C‐terminus).
  4. HeLa cells expressing luminal SI‐GFP chimeras of sucrose‐isomaltase (SI‐GFP, green) containing the WT and mutant N‐terminal cytosolic tails of LCS were fixed and labelled for surface localised GFP (red). Cells expressing LCS‐SI‐RxR and LCS‐SI‐LxxR chimeras show PM staining. Scale bar, 20 μm, (left). PM (surface) fluorescence intensity (in Appendix Table S10 are listed the software used in this study) of each chimera is normalised on the LCS‐SI‐GFP surface intensity (data are means ± SEM derived from two biological replicates; n is indicated in the graph, ***P < 0.001 [Student’s t‐test]) (right).
  5. Quantitative flow cytometry‐based analysis of the experiment reported in (D); n is indicated in the graph (left). Percentage of cells with plasma membrane staining (surface) is reported for each chimera (right).
  6. GOLPH3‐KD or OE HeLa cells were processed for ShTxB (upper panels) or ChTxB (lower panels) staining followed by flow cytometry analysis. Flow cytometry distributions and the relative scatter plots reporting the percentage of positive cells are shown for each condition.

Source data are available online for this figure.