Abstract
Although vesicular transport of the H-Ras protein from the Golgi to the plasma membrane is well known, additional trafficking steps, both to and from the plasma membrane, have also been described. Notably, both vesicular and nonvesicular transport mechanisms have been proposed. The initial trafficking of H-Ras to the plasma membrane was therefore examined in more detail. In untreated cells, H-Ras appeared at the plasma membrane more rapidly than a protein carried by the conventional exocytic pathway, and no H-Ras was visible on Golgi membranes in >80% of the cells. H-Ras was still able to reach the plasma membrane when COP II-directed transport was disrupted by two different mutant forms of Sar1, when COP I-mediated vesicular traffic from the endoplasmic reticulum to the Golgi was inhibited with brefeldin A, or when microtubules were disrupted by nocodazole. Although some H-Ras was present in the secretory pathway, protein that reached the membranes of the endoplasmic reticulum-Golgi intermediate compartment was unable to move further in the presence of nocodozale. These results identify an alternative mechanism for H-Ras trafficking that circumvents conventional COPI-, COPII-, and microtubule-dependent vesicular transport. Thus, H-Ras has two simultaneous but distinct means of transport and need not depend on vesicular trafficking for its delivery to the plasma membrane.
Highlights
Plasma membrane [3, 8, 9]
The COP II vesicles that are released from the widely dispersed ER exit site (ERES) seem to merge into a perinuclear vesiculo-tubular center sometimes termed the ER-Golgi-intermediate compartment (ERGIC)
The results indicate that two distinct routes for H-Ras transport operate simultaneously, and that a nonconventional, COP I- and COP II-independent mechanism moves the bulk of H-Ras to the plasma membrane
Summary
Plasmids, and Antibodies—H-Raswt, H-RasQ61L, and GFP-H-Ras were expressed from pcDNA3 (Invitrogen). Counting Cells—NIH 3T3 cells were co-transfected with cDNAs for YFP-GT46 and H-Ras, as indicated. Brefeldin A, Nocodazole, and Cycloheximide Treatment—At 6 h after transfection, brefeldin A (BFA, 5 g/ml) was applied to the cells in regular medium. Cells were fixed and visualized by immunofluorescence at 30, 60, and 90 min after BFA treatment. Cells were treated with cycloheximide (50 g/ml) immediately after the transfection. After 6 h the cycloheximide was washed out, and fresh medium with either BFA or nocodazole (20 g/ml) was added. Cells were transfected, treated with cycloheximide for 6 h, and were rinsed to wash out the cycloheximide, and nocodazole was applied for 4 h. Immunofluorescence Imaging—NIH 3T3 and COS-7 cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) at room temperature for 15 min. The contrast and signal strength of images were balanced, and images were deconvolved and merged as indicated, using Improvision OpenLab and Adobe Photoshop software
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