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Specific interactions of Mss4 with members of the Rab GTPase subfamily
J L Burton1, M E Burns, E Gatti
1Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510.
Abstract:
Mss4 is a mammalian protein that was identified as a suppressor of a yeast secretory mutant harboring a mutation in the GTPase Sec4 and was found to stimulate GDP release from this protein. We have now performed a biochemical characterization of the Mss4 protein and examined the specificity of its association with mammalian GTPases. Mss4 is primarily a soluble protein with a widespread tissue distribution. Recombinant Mss4 binds GTPases present in tissue extracts, and by a gel overlay assay binds specifically Rab Rab10proteins. We further define the Mss4-GTPase interaction to a subset of Rabs belonging to the same subfamily branch which include Rab1, Rab3, Rab8, Rab10, Sec4 and Ypt1 but not Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11. Accordingly, Mss4 co-precipitates from a brain extract with Rab3a but not Rab5. Mss4 only stimulates GDP release from, and the association of GTP gamma S with, this Rab subset. Recombinant Mss4 and Rab3a form a stable complex in solution that is dissociated with either GDP or GTP gamma S. Injection of Mss4 into the squid giant nerve terminal enhances neurotransmitter release. These results suggest that Mss4 behaves as a guanylnucleotide exchange factor (GEF) for a subset of Rabs to influence distinct vesicular transport steps along the secretory pathway.
Insights
Mss4 is a guanine nucleotide exchange factor (GEF) that regulates vesicular transport. It specifically activates a subset of Rab GTPases, influencing key steps in the secretory pathway.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mss4, a mammalian protein, was initially identified as a suppressor of a yeast secretory mutant.
- The yeast protein Sec4, a GTPase, was involved in the mutation.
Purpose of the Study:
- To biochemically characterize Mss4 protein.
- To investigate the specificity of Mss4's interaction with mammalian GTPases.
- To understand Mss4's role in vesicular transport.
Main Methods:
- Biochemical characterization of Mss4.
- Gel overlay assay to determine binding specificity.
- Co-precipitation assays using brain extracts.
- In vitro assays to measure GDP release and GTP gamma S binding.
- Functional assay involving injection into squid giant nerve terminals.
Main Results:
- Mss4 is a soluble protein with broad tissue distribution.
- Mss4 specifically binds and stimulates GDP release from a subset of Rab GTPases (Rab1, Rab3, Rab8, Rab10, Sec4, Ypt1).
- Mss4 forms a stable complex with Rab3a, dissociable by GDP or GTP gamma S.
- Mss4 enhances neurotransmitter release when injected into squid giant nerve terminals.
Conclusions:
- Mss4 functions as a guanine nucleotide exchange factor (GEF) for a specific subset of Rab GTPases.
- Mss4 plays a role in regulating distinct steps of vesicular transport within the secretory pathway.