Figure 7. Prosaposin N-terminus region... | Rockefeller University Press

Figure 7.

$Prosaposin N-terminus region is required for interaction with Surf4 and ER exit.(A) Organization of prosaposin showing saposin domains A–D in rectangles. The blue area indicates the signal sequence of prosaposin, and dashed red lines indicate sites of N- and C-terminal deletions. (B–D) Live-cell imaging of prosaposin-RUSH traffic from ER to Golgi of WT, N-terminal Δ mutant, and C-terminal Δ mutant; scale bar, 10 µm. Magnified insets show the localization of prosaposin to GFP-GalT–labeled Golgi; inset scale bar, 2 µm. (E) Time-dependent delivery of prosaposin-RUSH traffic to the Golgi (defined by GFP-GalT signal) for prosaposin-WT, N-terminal Δ mutant, and C-terminal Δ mutant. The mean fluorescence intensities of prosaposin-RUSH localized to the Golgi at each time point after biotin addition were normalized to the mean fluorescence intensities before biotin (baseline marked by gray line). Data were collected from three independent experiments with n = 9–12 cells per experiment; error bars show mean ± SEM. (F) Immunoblots from anti-GFP-Surf4 IPs show a reduction of the N-terminally deleted prosaposin (N-terminal Δ mutant) in the IP fraction of GFP-Surf4, in comparison with C-terminal Δ mutant of prosaposin. Note that while the overall levels of the prosaposin C-terminal Δ mutant are lower than the WT, the proportion that interacts with Surf4 is similar. (G) Quantification of the ratio of prosaposin-RUSH to GFP-Surf4 in IP fractions. Prosaposin-RUSH levels in IP fractions were normalized to input samples. n = 3 independent experiments; error bars show mean ± SEM.$

Prosaposin N-terminus region is required for interaction with Surf4 and ER exit. (A) Organization of prosaposin showing saposin domains A–D in rectangles. The blue area indicates the signal sequence of prosaposin, and dashed red lines indicate sites of N- and C-terminal deletions. (B–D) Live-cell imaging of prosaposin-RUSH traffic from ER to Golgi of WT, N-terminal Δ mutant, and C-terminal Δ mutant; scale bar, 10 µm. Magnified insets show the localization of prosaposin to GFP-GalT–labeled Golgi; inset scale bar, 2 µm. (E) Time-dependent delivery of prosaposin-RUSH traffic to the Golgi (defined by GFP-GalT signal) for prosaposin-WT, N-terminal Δ mutant, and C-terminal Δ mutant. The mean fluorescence intensities of prosaposin-RUSH localized to the Golgi at each time point after biotin addition were normalized to the mean fluorescence intensities before biotin (baseline marked by gray line). Data were collected from three independent experiments with n = 9–12 cells per experiment; error bars show mean ± SEM. (F) Immunoblots from anti-GFP-Surf4 IPs show a reduction of the N-terminally deleted prosaposin (N-terminal Δ mutant) in the IP fraction of GFP-Surf4, in comparison with C-terminal Δ mutant of prosaposin. Note that while the overall levels of the prosaposin C-terminal Δ mutant are lower than the WT, the proportion that interacts with Surf4 is similar. (G) Quantification of the ratio of prosaposin-RUSH to GFP-Surf4 in IP fractions. Prosaposin-RUSH levels in IP fractions were normalized to input samples. n = 3 independent experiments; error bars show mean ± SEM.