Key Points
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H-ras and N-ras traffic through the Golgi to the plasma membrane, whereas K-ras traffics through the cytosol.
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H-ras and K-ras localize to spatially distinct microdomains of the plasma membrane, yielding new insights into plasma-membrane microstructure.
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H-ras and K-ras interaction with these microdomains is dynamic and in the case of H-ras is regulated by GTP-loading. Signal output from these different microdomains also varies.
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N-ras and H-ras are stably expressed on Golgi membranes in addition to plasma membrane. H-ras and K-ras have also been identified on endosomal membranes.
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Ras is activated on Golgi membranes in response to cell-surface growth-factor activation.
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Ras proteins on endomembranes have distinct signal outputs that might contribute to their isoform-specific functions.
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Biological differences between the Ras proteins can probably be explained by their distinct membrane microenvironments.
Abstract
Ras signalling has classically been thought to occur exclusively at the inner surface of a relatively uniform plasma membrane. Recent studies have shown that Ras proteins interact dynamically with specific microdomains of the plasma membrane as well as with other internal cell membranes. These different membrane microenvironments modulate Ras signal output and highlight the complex interplay between Ras location and function.
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Acknowledgements
I would like to thank R. Parton and I. Prior for their helpful comments on the manuscript, and M. Philips and A. Pellicer for sharing data before publication. J.F.H. is supported by grants from the National Health and Medical Research Council, Australia, and the Queensland Cancer Fund. The Institute for Molecular Bioscience is a special research centre of the Australian Research Council.
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Glossary
- HYPERVARIABLE DOMAIN
-
The carboxy-terminal 25 amino acids of H-ras, N-ras and K-Ras proteins, in which sequence homology is less than 15% between any two isoforms, compared with 90–100% over the amino-terminal sequences.
- CAAX MOTIF
-
(where C is cysteine, A is aliphatic and X is any amino acid). A carboxy-terminal tetrapeptide that is common to all Ras proteins. It directs a triplet of post-translational modifications.
- FRET
-
(fluorescence resonance energy transfer). The fluorescence energy that is transferred from one fluor excites a neighbouring fluor that then re-emits the energy at a third wavelength. Transfer occurs only if the two fluors are close, so FRET can be used to monitor real-time protein–protein interactions in living cells.
- PHOTOBLEACHING
-
The irreversible destruction, by any one of several different mechanisms, of a fluorophore that is under illumination.
- FRAP
-
(fluorescence recovery after photobleaching). The time taken for a bleached area of membrane to re-fluoresce. FRAP can be used to calculate the lateral mobility of a green fluorescent protein (GFP)-tagged membrane-associated protein on the condition that recovery does not occur by exchange with a soluble pool of protein.
- MICRODOMAINS
-
Sites within the plasma membrane that have a distinct lipid and/or protein composition.
- PALMITOYLATION
-
The post-translational modification of a protein with palmitic acid. On Ras proteins, palmitic acid is attached as a thioester to the thiol (–SH) group of cysteine residues (this is known as S-acylation).
- BREFELDIN A
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A reagent that is used to disassemble the Golgi, probably by inhibiting the GTP-loading of ADP-ribosylation factor. In sensitive cells the Golgi is dispersed and trafficking of proteins through the exocytic pathway is inhibited.
- DOMINANT-NEGATIVE
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A defective protein that retains interaction abilities and so distorts or competes with normal proteins.
- RHO FAMILY PROTEINS
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Ras-related GTPases that are involved in controlling the polymerization of actin.
- TAXOL
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An antitumour agent that enhances the polymerization of tubulin and the subsequent stabilization of microtubules, thereby inhibiting mitosis and blocking the cell cycle.
- LIPID RAFTS
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The biophysical properties of certain long-chain saturated fatty acids packed together with cholesterol drive the spontaneous formation of small, relatively stable, structures that are known as lipid rafts. Lipid rafts phase separate from the more loosely packed phospholipids of the membrane bilayer.
- LIQUID-ORDERED
-
A term that is used to describe the tightly packed molecular structure of a lipid raft: an intermediate physical state between fluid and gel.
- DISORDERED PLASMA MEMBRANE
-
Plasma membrane that is not organized in liquid-ordered lipid rafts.
- RAB PROTEINS
-
Rab proteins form the largest subfamily of small GTPases of the Ras superfamily. They regulate budding, tethering, fusion and motility at various sites within cells.
- YEAST TWO-HYBRID APPROACH
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A technique that is used to test if two proteins physically interact with each other. One protein is fused to the GAL4 activation domain and the other to the GAL4 DNA-binding domain, and both fusion proteins are introduced into yeast. Expression of a GAL4-regulated reporter gene indicates that the two proteins physically interact.
- PC12 CELLS
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A clonal line of rat adrenal pheochromocytoma cells that, much like sympathetic neurons, respond to nerve growth factor and can synthesize, store and secrete catecholamines. PC12 cells contain small, clear synaptic-like vesicles and larger dense core granules.
- COS CELLS
-
Cells from the monkey CV1 cell line that have an integrated SV40 genome lacking an origin of replication. Plasmids with an SV40 origin of replication are replicated to a high copy number when transfected.
- ENDOMEMBRANE SYSTEM
-
A hypothetical integrated membrane system of eukaryotic cells that represents a developmental and functional continuum. It comprises the endoplasmic reticulum, nuclear membrane, Golgi apparatus and vesicles.
- NIH3T3 FOCUS ASSAY
-
This assay measures the transforming potency of an oncogene. The growth of NIH3T3 cells arrests as a continuous monolayer, but transformed cells do not arrest and grow over the adjacent monolayer as a focus of clonal cells.
- EEA1
-
The antigen that is involved in a human autoimmune disease. It is a marker of the early endosome.
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Hancock, J. Ras proteins: different signals from different locations. Nat Rev Mol Cell Biol 4, 373–385 (2003). https://doi.org/10.1038/nrm1105
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DOI: https://doi.org/10.1038/nrm1105