Quantitative Identification of the Haloferax volcanii Secretome
EMSL Project ID
47650
Abstract
To carry out numerous cellular processes of fundamental importance, prokaryotes must have efficient means of secreting proteins into the extracytoplasmic environment, including proteins involved in the degradation of polymers, the uptake of nutrients, generation of energy and the formation and maintenance of biofilms. Two important pathways that play particularly crucial roles in the transport across the cytoplasmic membranes are the universally conserved Sec pathway and the more recently identified Tat pathway. After translocation and cleavage, pathway substrates often undergo additional post-translational modifications, and although these modifications are often essential to the stability and function of the secreted protein, key aspects of these modifications appear to be taxon specific. For example, lipoboxes at the C-terminus of a signal peptide are essential for successful lipoprotein processing and protein membrane-anchoring via lipids. Yet archaeal homologs of bacterial lipoprotein biosynthesis components have not been identified, nor have mechanisms or components specific to archaeal lipid modification. Other apparent domain specific modifications include the methylation of bacterial type IV pili and the N-glycosylation of archaeal type IV pili.In this study, we propose to use topdown mass spectrometry to identify and characterize the secretome of Haloferax volcanii, a model archaeon adapted to growth at high salt concentrations. Haloarchaea are particularly suited as model systems for characterizing archaeal secretomes as they have well-characterized Sec substrates, and extensively use the Tat pathway to transport secreted proteins, as well as lipid-anchoring of these substrates to the cell membrane. The specific aims of the proposed research are: 1) to identify the Sec and Tat substrates from supernatant and membrane protein fractions to the fullest extent possible using topdown proteomics; and 2) to characterize the post-translational modifications of these secreted proteins.
These studies will provide invaluable data about the expression, subcellular localization and signal peptide processing, as well as lipid and sugar modifications of Sec and Tat substrates. Moreover, these analyses may identify substrates that are not predicted to be transported via either of these pathways and therefore have the potential to reveal additional, currently unknown, secretion pathways. Since haloarchaea are a type of extremophile, these analyses will also aid in characterizing the boundaries and limitations of biological processes in the biosphere. We expect that the analyses of the results generated by this project will reveal fundamental insights into protein transport that will allow the development of strategies that result in an increased efficiency of transport and post-translational modification of proteins. We request rapid access to EMSL on this project to gather preliminary data in preparation for submitting a joint collaborative NSF grant next January. A proposal is currently nearly complete, primarily lacking the preliminary data. We anticipate that the data generated under this scope of work will identify many exciting discoveries for archaeal cell biology and protein secretion pathways. This data will serve as preliminary data and a solid justification for our future work exploring new secretion mechanisms.
Project Details
Project type
Limited Scope
Start Date
2012-12-07
End Date
2013-02-06
Status
Closed
Released Data Link
Team
Principal Investigator
Related Publications
Halim MF, F Pfeiffer, J Zou, A Frisch, DH Haft, S Wu, N Tolic, HM Brewer, SH Payne, L Pasa-Tolic, and M Pohlschroder. 2013. "Haloferax volcanii archaeosortase is required for motility, mating, and C-terminal processing of the S-layer glycoprotein." Molecular Microbiology 88(6):1164-1175. doi: 10.1111/mmi.12248