Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Jul 18;170(2):261-72.
doi: 10.1083/jcb.200502063.

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

J Shawn Goodwin  1 , Kimberly R Drake, Carl Rogers, Latasha Wright, Jennifer Lippincott-Schwartz, Mark R Philips, Anne K Kenworthy
Affiliations

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

J Shawn Goodwin et al. J Cell Biol. .

Abstract

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
GFP-CAAX proteins and GFP-Ras palmitoylation mutants traffic rapidly between the ER and Golgi complex. (A) The Golgi-associated pool of a GFP-HRas C181S, C184S recovers rapidly and completely within 1 min after photobleaching of the entire Golgi complex (circle, t = 0). Bar, 10 μm. (B) Kinetics of fluorescence recovery after bleaching the entire Golgi-associated pool of GFP-CVLS (circles), GFP-CLLL (closed squares), GFP-HRas C181S, C184S (open squares), and GFP-NRas C181S (triangles). Data are from a representative experiment. For clarity, error bars are not shown. (C) Halftimes of fluorescence recovery for Golgi photobleaching experiments. Data are the mean ± SE from four independent experiments (∼10 cells per experiment). Golgi bleaches were performed at 22°C.
Figure 2.
Figure 2.
Diffusional mobilities of GFP-CAAX proteins and GFP-Ras palmitoylation mutants in the ER measured by confocal FRAP. (A) Representative images from a confocal FRAP measurement of the lateral diffusion of GFP-NRasC181S in the ER. The bleach box (white) is 4 μm deep. Recovery occurs so rapidly that it is difficult to see the bleached region at the 6.7-s time point. Bar, 10 μm. (B) Recovery curves from ER-associated pools of GFP-CVLS (circles), GFP-CLLL (squares), GFP-HRas C181S, C184S (diamonds), and GFP-NRas C181S (crosses); t = 22°C. Data are from a representative experiment (n = 10 cells/protein; mean ± SE). For comparison, a recovery curve for GFP-HRas at the cell surface is shown (triangles). (C) D for ER-associated protein calculated from confocal FRAP experiments. D for GFP-HRas at the cell surface is shown for comparison.
Figure 3.
Figure 3.
FCS detects both a soluble and membrane-associated pool of GFP-CAAX proteins and GFP-Ras palmitoylation mutants. (A) Normalized FCS autocorrelation curves for GFP-CVLS (circles), GFP-CLLL (squares), GFP-HRas C181S, C184S (diamonds), and GFP-NRasC181S (dotted line) measured in the ER. As a control, measurements were also made for cytosolic GFP (inverted triangle) under similar conditions. Each curve is the average of 10 measurements on an individual cell from a representative experiment. (B) A two-component model (red) rather than a one-component model (blue) is required to describe the autocorrelation data for GFP-CVLS. The τD for the fast component (174 μs) is similar to free GFP, whereas τD for the slower component (3,195 μs) corresponds to D of 2.3 μm2/s. The inset shows a plot of the residuals for each fit. FCS data were obtained at 22°C.
Figure 4.
Figure 4.
GFP-NRas and GFP-HRas are trafficked to the Golgi complex in the absence of new protein synthesis under steady-state conditions. (A) GFP-NRas is localized to the Golgi complex in the absence of new protein synthesis. Cells were either mock treated (control) or incubated in the presence of 200 μg/ml cycloheximide (CHX) for 4 h at 37°C before imaging. For comparison, the distribution of GFP-GPI, a protein that cycles between the Golgi complex and cell surface, is shown. Bar, 10 μm. (B) The Golgi-associated pool of GFP-NRas partially recovers after photobleaching of the entire Golgi compartment (white circles, t = 0). Cells were treated with cycloheximide for 4 h before the experiment. Bar, 10 μm. (C) Kinetics of recovery of fluorescence in the Golgi complex for GFP-HRas (closed squares) and GFP-NRas (open circles) at 37°C (n = 8–9 cells). Note the differences in the extent of recovery for the two proteins. Data are from a representative experiment. Similar results were obtained in the presence or absence of cycloheximide. Bars = SE. (D) Kinetics of Golgi recovery for GFP-NRas (open circles) versus GFP-GPI (closed squares) at 37°C (n = 6–9 cells). Bars = SE.
Figure 5.
Figure 5.
Recycling of GFP-HRas and GFP-NRas to the Golgi complex occurs by a microtubule-independent pathway. (A) Cells expressing GFP-NRas were subjected to acute nocodazole treatment as described in the Materials and methods before imaging. The Golgi-associated pool of GFP-NRas was then selectively photobleached (circle, t = 0) and fluorescence recovery was monitored over time. Bar, 10 μm. (B) Acute nocodazole treatment (NZ) inhibits delivery of Cy3-CTXB to the perinuclear region after 20 min of internalization, a time point at which significant Cy3-CTXB accumulation is observed in the perinuclear region in cells treated with vehicle alone (control). Bar, 10 μm. (C and D) Kinetics of recovery of GFP-HRas (C) and GFP-NRas (D) to the Golgi complex are not significantly altered by acute nocodazole treatment. Data are from a representative experiment at 37°C. Bars = SE.
Figure 6.
Figure 6.
2BP inhibits delivery of GFP-HRas and GFP-NRas, but not YFP-GL-GPI, to the cell surface. COS-7 cells were treated with 2BP or vehicle overnight immediately after transfection with (A) GFP-HRas, (B) GFP-NRas, or (C) YFP-GL-GPI and imaged the following day. Bars, 10 μm.
Figure 7.
Figure 7.
Time course of redistribution of GFP-HRas and GFP-NRas in response to 2BP treatment. (A) Cells were transfected with GFP-HRas or GFP-NRas, allowed to express the protein overnight, and then treated with 2BP for the indicated times before imaging. Bars, 10 μm. (B) Cells treated with 2BP were scored for ER labeling as described in Materials and methods. Data show the mean ± SE for four independent experiments. (C) Cells treated with 2BP were scored for the fraction of Ras localized to the Golgi complex as described in the Materials and methods. Data are the mean ± SE from three independent experiments.
Figure 8.
Figure 8.
Working model for how palmitoylation regulates HRas and NRas trafficking to and from the Golgi complex. (1) Before palmitoylation, newly synthesized Ras can reversibly bind ER and Golgi membranes and traffic between them via a soluble cytosolic intermediate. (2) Palmitoylation via a putative palmitoyl acyl transferase (PAT) kinetically traps Ras onto membranes in the early secretory pathway, and (3) enables the protein to be packaged into vesicles bound for the cell surface. Once reaching the cell surface, palmitoylated HRas and NRas can undergo endocytosis (not depicted). (4) Turnover of palmitate generates a transiently depalmitoylated pool of protein that is returned to the Golgi complex and/or ER by nonvesicular transport, where it can again interact with PAT and reenter the secretory pathway.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Apolloni, A., I.A. Prior, M. Lindsay, R.G. Parton, and J.F. Hancock. 2000. H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Mol. Cell. Biol. 20:2475–2487. - PMC - PubMed
    1. Baker, T.L., M.A. Booden, and J.E. Buss. 2000. S-nitrosocysteine increases palmitate turnover on Ha-Ras in NIH 3T3 cells. J. Biol. Chem. 275:22037–22047. - PubMed
    1. Baker, T.L., H. Zheng, J. Walker, J.L. Coloff, and J.E. Buss. 2003. Distinct rates of palmitate turnover on membrane-bound cellular and oncogenic H-ras. J. Biol. Chem. 278:19292–19300. - PubMed
    1. Berthiaume, L., and M.D. Resh. 1995. Biochemical characterization of a palmitoyl acyltransferase activity that palmitoylates myristoylated proteins. J. Biol. Chem. 270:22399–22405. - PubMed
    1. Bivona, T.G., and M.R. Philips. 2003. Ras pathway signaling on endomembranes. Curr. Opin. Cell Biol. 15:136–142. - PubMed

Publication types

MeSH terms