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. 2010 Oct 4;191(1):23-9.
doi: 10.1083/jcb.200911143. Epub 2010 Sep 27.

Palmitoylated Ras proteins traffic through recycling endosomes to the plasma membrane during exocytosis

Ryo Misaki  1 , Miki Morimatsu, Takefumi Uemura, Satoshi Waguri, Eiji Miyoshi, Naoyuki Taniguchi, Michiyuki Matsuda, Tomohiko Taguchi
Affiliations

Palmitoylated Ras proteins traffic through recycling endosomes to the plasma membrane during exocytosis

Ryo Misaki et al. J Cell Biol. .

Abstract

Ras proteins regulate cell growth, death, and differentiation, and it is well established that this functional versatility is accomplished through their different subcellular localizations. Palmitoylated H- and N-Ras are believed to localize at the perinuclear Golgi and plasma membrane (PM). Notably, however, recycling endosomes (REs) also localize to a perinuclear region, which is often indistinguishable from the Golgi. In this study, we show that active palmitoylated Ras proteins mainly localize intracellularly at REs and that REs act as a way station along the post-Golgi exocytic pathway to the PM. H-Ras requires two palmitoyl groups for RE targeting. The lack of either or both palmitoyl groups leads to the mislocalization of the mutant proteins to the endoplasmic reticulum, Golgi apparatus, or the PM. Therefore, we demonstrate that palmitoylation directs Ras proteins to the correct intracellular organelles for trafficking and activity.

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Figures

Figure 1.
Figure 1.
Steady-state distribution of palmitoylated Ras proteins at REs. (A) Schematic illustration of organelle distribution in COS-1 cells. Golgi exhibits a ring-shape appearance (Golgi ring), and REs are confined within Golgi ring. (B–L) The indicated proteins (B, D, E, G, and H, GFP–H-Ras; F, RFP–H-Ras and GFP-VAMP3; I, RFP-VAMP3 and GFP–H-Ras; C and J, GFP–N-Ras; K, GFP–H-Ras–C20; L, GFP–N-Ras–C20) were expressed overnight in COS-1 (B–G and J–L), MDCK, or HeLa cells (H and I). Cells were treated with (D–L) or without (B and C) CHX for 4 h. Cells in G were pulsed for 20 min with transferrin–Alexa Fluor 546 and chased for 10 min to highlight REs. GM130 was immunostained to localize Golgi when necessary. The arrowheads indicate PM. Bars, 10 µm.
Figure 2.
Figure 2.
The essential role of palmitoyl groups of H-Ras in RE targeting. (A) GFP–H-Ras was expressed overnight. Cells were treated with 25 µM 2BP in the presence of CHX for the indicated times followed by fixation to immunostain GM130. (B) Transferrin–Alexa Fluor 546 was endocytosed to COS-1 cells, which had been treated with 2BP for 16 h. Cells were fixed to immunostain GM130. (C–F) The indicated palmitoyl-deficient mutant proteins (C, GFP–H-Ras–C181-184S; D, GFP–H-Ras–C181S; E, GFP–H-Ras–C184S; F, GFP–H-Ras–C184L) were expressed overnight. Cells were treated with CHX for 4 h followed by fixation to immunostain GM130. The arrowhead indicates PM. Bars, 10 µm.
Figure 3.
Figure 3.
Post-Golgi trafficking of H-Ras through REs to PM. (A and E) GFP–H-Ras and GFP–H-Ras–C184S expressed overnight are shown in A and E, respectively. Cells were subjected to 20°C block for 2 h with CHX. 20°C block was released with CHX for the indicated times followed by fixation to immunostain GM130. (right) Magnified images of boxed areas around the Golgi/REs area are shown. Bars, 10 µm. The arrowheads indicate PM. (B) Circles represent the Pearson coefficient between GFP–H-Ras and GM130 from the individual cells shown in A at 0 (n = 50) or 120 min (n = 50). *, P < 0.001. Error bars indicate mean ± SD. (C) GFP–H-Ras and RFP-Rab6a were coexpressed overnight. Cells were subjected to 20°C block for 2 h with CHX. Immediately upon shifting from 20 to 37°C, the Golgi/REs area was imaged every 10 min for 60 min. Bar, 5 µm. (D) GFP intensity in C was measured every 10 min in two areas: (1) Golgi plus REs (blue) and (2) REs (red). The results of four independent experiments are shown with different symbols. Individual GFP intensities are normalized to the GFP intensity from Golgi plus REs at 0 min.
Figure 4.
Figure 4.
Exocytic pathway of palmitoylated Ras proteins through REs to PM. The sequences of C-terminal 20–amino acid stretch of Ras proteins and palmitoylation deficient mutants used in this study are shown. After the exit from Golgi, H-Ras is first transported to REs, then to PM. A palmitoyl group on C181 (green) is required to exit Golgi. Without the palmitoyl group on C184 (blue) upon the exit, mutant H-Ras (H-Ras–C184S) is directly transported to PM. L184 can substitute the palmitoyl group on C184 (H-Ras–C184L can access REs), which explains N-Ras residency on REs.
Figure 5.
Figure 5.
H-Ras is active at REs. (A–G) The indicated proteins (A, GFP-H–Ras; B, GFP–H-Ras–C181S; C, GFP–H-Ras–C184S; D, GFP–H-RasG12V; E, GFP–H-RasG12V–C181S; F, GFP–H-RasG12V–C184S; G, GFP–H-Ras–C20) were expressed overnight together with RFP-RBD. Cells were treated with CHX for 4 h. A fluorescence intensity line scan profile was generated along the yellow line, which goes through the Golgi/REs area and shown is in the right column. The arrowheads indicate PM. Bars, 10 µm.
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