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. 2024 Jun 5;12:RP89306. doi: 10.7554/eLife.89306

Figure 3. Golgi exit kinetics of LAT are dependent on its association with raft domains.

(A) Representative confocal images of Golgi RUSH experiment show notable Golgi retention of non-raft constructs (LAT-allL and allL-TMD) after 90 min of biotin addition, in contrast to raft-preferring LAT and LAT-TMD. (B) Temporal reduction of protein constructs remaining in Golgi after biotin addition (i.e. release from Golgi RUSH), quantified by immunostaining and colocalization with Golgi marker (Giantin, see Figure 3—figure supplement 2). Symbols represent average +/-st.dev. from three independent experiments with >15 cells/experiment. Fits represent exponential decays; shading represents 95% confidence intervals. (C) Golgi exit rates for raft-associated LAT constructs are ~2.5-fold faster than non-raft versions for both full-length and TMD-only. Points represent t1/2 values from fits of independent repeats with >20 cells/experiment. *p<0.05, nsp >0.05. (D) Representative confocal images of full-length LAT and LAT-allL during Golgi temperature block. Addition of biotin at 17 °C releases ER-RUSH constructs but traps them in Golgi. Removing temperature block by incubation at 37 °C leads to fast PM trafficking of LAT but not non-raft LAT-allL. (E) Fraction of proteins in Golgi for the constructs shown in D, calculated as in B. Fits represent exponential decays. (inset) Golgi exit kinetics quantified as in C. All scale bars = 5 μm. Original data quantification can be found in the Source Data files.

Figure 3—source data 1. Quantification of the temporal reduction of protein constructs remaining in Golgi after biotin addition.
Figure 3—source data 2. Quantification of Golgi exit rates.
Figure 3—source data 3. Quantification of the time residency of constructs in Golgi calculated from the kinetics.
Figure 3—source data 4. Quantification of the temporal kinetics after temperature block in the Golgi.

Figure 3.

Figure 3—figure supplement 1. Fraction of RUSH-VSVG in Golgi after biotin addition.

Figure 3—figure supplement 1.

Inset represents repeats quantifications of the half-time of Golgi exit. Symbols represent average +/-st.dev. from three independent experiments.
Figure 3—figure supplement 2. Example of quantification of Golgi residence for protein of interest (POI).

Figure 3—figure supplement 2.

Top panels are representative images, bottom panels are corresponding masks to calculate the fraction of POI in Golgi. Giantin was used as Golgi marker to create the mask for that organelle. Cells mask represents the cell border from the POI channel after background subtraction.