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Human rabaptin-5 is selectively cleaved by caspase-3 during apoptosis
E Swanton1, N Bishop, P Woodman
1School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Abstract:
We have previously shown that Xenopus rabaptin-5 is cleaved in apoptotic extracts, with a concomitant reduction in the ability of these extracts to support endosomal membrane fusion (Cosulich, S. C., Horiuchi, H., Zerial, M., Clarke, P. R., and Woodman, P. G. (1997) EMBO J. 16, 6182-6191). In this report we demonstrate that caspase-dependent cleavage is a conserved feature of rabaptin-5. Human rabaptin-5 is cleaved at two sites (HSLD(379) and DESD(438)) in apoptotic HeLa extracts. Cleavage is effected by caspase-3, since it is prevented when caspase-3 activity is either inhibited by Ac-DEVD-CHO or removed by immunodepletion. Moreover, an identical pattern of cleavage is observed using recombinant caspase-3. The action of caspase-3 is highly selective; neither caspase-2 nor caspase-7 are able to cleave recombinant or cytosolic rabaptin-5. Caspase-dependent cleavage of rabaptin-5 generates two physically separated coiled coil-forming domains, the C-terminal of which retains the ability to bind the Rab5 exchange factor rabex-5.
Insights
Caspase-3 selectively cleaves human rabaptin-5 during apoptosis, generating domains that bind rabex-5. This conserved cleavage impacts endosomal membrane fusion.
Area of Science:
- Cell biology
- Molecular biology
- Apoptosis research
Background:
- Xenopus rabaptin-5 cleavage in apoptotic extracts reduces endosomal membrane fusion.
- Rabaptin-5 plays a role in endosomal trafficking and membrane fusion.
Purpose of the Study:
- To investigate the conservation of rabaptin-5 cleavage in human apoptotic cells.
- To identify the specific caspase responsible for rabaptin-5 cleavage.
- To determine the functional consequences of rabaptin-5 cleavage.
Main Methods:
- Analysis of human rabaptin-5 cleavage in apoptotic HeLa cell extracts.
- Use of caspase-3 inhibitors (Ac-DEVD-CHO) and immunodepletion.
- Cleavage assays with recombinant caspase-3, caspase-2, and caspase-7.
- Characterization of cleaved rabaptin-5 domains and their binding properties.
Main Results:
- Human rabaptin-5 is cleaved at two specific sites (HSLD(379) and DESD(438)) in apoptotic HeLa extracts.
- Caspase-3 is the primary enzyme responsible for rabaptin-5 cleavage; caspases-2 and -7 do not cleave rabaptin-5.
- Cleavage by caspase-3 generates two distinct domains, with the C-terminal domain retaining binding to rabex-5.
Conclusions:
- Caspase-dependent cleavage of rabaptin-5 is a conserved event in apoptosis.
- Caspase-3 selectively targets rabaptin-5, impacting its function in endosomal trafficking.
- The C-terminal domain of cleaved rabaptin-5 maintains interaction with rabex-5, suggesting a role in regulating Rab5-mediated endocytosis during apoptosis.