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Association of the Ras-antagonistic Rap1/Krev-1 proteins with the Golgi complex
F Béranger1, B Goud, A Tavitian
1Institut National de la Santé et de la Recherche Médicale, Unité 248, Faculté de Médecine, Paris, France.
Abstract:
Ras oncogenes encode 21-kDa GTP-binding proteins that are capable of transforming immortalized cells in culture. Ras proteins are bound to the inner face of the plasma membrane by their C-terminal extremity and are thought to transmit their mitogenic signals via an "effector" domain spanning amino acids 32-42. Two ras-related human genes rap1A and rap1B encode 95% homologous 21-kDa proteins that share with Ras p21 the same effector domain and a similar C-terminal Cys-Ali-Ali-Xaa sequence (where Ali is an aliphatic amino acid; also known as a CAAX sequence). The product of the rap1A gene is identical to that of the Krev-1 cDNA, whose overexpression is capable of reverting the phenotype of Ki-ras-transformed NIH 3T3 cells. Antibodies that do not cross-react with Ras and other Ras-related proteins were obtained by immunizing rabbits with a peptide encompassing residues 121-137 of Rap1 proteins. These antibodies were used to investigate the subcellular localization of Rap1 proteins by indirect immunofluorescence and fractionation techniques. Rap1 proteins were found to be tightly bound to cellular membranes. They did not colocalize with Ras proteins on the plasma membrane and were discovered to be associated with the Golgi complex.
Insights
Ras-related proteins Rap1A and Rap1B are membrane-bound and localize to the Golgi complex, distinct from Ras proteins on the plasma membrane. This finding offers new insights into cellular signaling pathways.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncogenes
Background:
- Ras oncogenes encode 21-kDa GTP-binding proteins crucial for cell transformation.
- Ras proteins are anchored to the plasma membrane and transmit mitogenic signals via an effector domain.
- Rap1A and Rap1B are highly homologous ras-related proteins sharing key structural features with Ras p21.
Purpose of the Study:
- To investigate the subcellular localization of Rap1 proteins.
- To determine if Rap1 proteins share the plasma membrane localization with Ras proteins.
- To understand the distinct cellular roles of Rap1 proteins in relation to Ras.
Main Methods:
- Generation of specific antibodies against Rap1 proteins (residues 121-137).
- Indirect immunofluorescence microscopy to visualize Rap1 protein localization.
- Cellular fractionation techniques to isolate membrane-bound proteins.
- Comparison of Rap1 localization with Ras protein localization.
Main Results:
- Rap1 proteins were found to be tightly associated with cellular membranes.
- Rap1 proteins did not colocalize with Ras proteins on the plasma membrane.
- Rap1 proteins were identified as being associated with the Golgi complex.
Conclusions:
- Rap1 proteins exhibit a distinct subcellular localization compared to Ras proteins.
- The Golgi complex is a key cellular compartment for Rap1 protein function.
- Understanding Rap1 localization provides insights into its role in cellular signaling and potential as a therapeutic target.