Diversity and subcellular distribution of archaeal secreted proteins

Abstract

Secreted proteins make up a significant percentage of a prokaryotic proteome and play critical roles in important cellular processes such as polymer degradation, nutrient uptake, signal transduction, cell wall biosynthesis, and motility. The majority of archaeal proteins are believed to be secreted either in an unfolded conformation via the universally conserved Sec pathway or in a folded conformation via the Twin arginine transport (Tat) pathway. Extensive in vivo and in silico analyses of N-terminal signal peptides that target proteins to these pathways have led to the development of computational tools that not only predict Sec and Tat substrates with high accuracy but also provide information about signal peptide processing and targeting. Predictions therefore include indications as to whether a substrate is a soluble secreted protein, a membrane or cell wall anchored protein, or a surface structure subunit, and whether it is targeted for post-translational modification such as glycosylation or the addition of a lipid. The use of these in silico tools, in combination with biochemical and genetic analyses of transport pathways and their substrates, has resulted in improved predictions of the subcellular localization of archaeal secreted proteins, allowing for a more accurate annotation of archaeal proteomes, and has led to the identification of potential adaptations to extreme environments, as well as phyla-specific pathways among the archaea. A more comprehensive understanding of the transport pathways used and post-translational modifications of secreted archaeal proteins will also facilitate the identification and heterologous expression of commercially valuable archaeal enzymes.

Keywords: Sec transport; Tat transport; archaea; archaeosortase; cell surface structures; lipoprotein; pili; protein transport.

Figure 1

Archaeal protein secretion and subcellular localization. Proteins that contain Tat signal peptides pass the cytoplasmic membrane (CM) through the Tat translocon after translation and folding. Conversely, protein translocation through the Sec pore can occur co- and possibly post-translationally. Upon secretion and signal peptide processing, Sec and Tat substrates can be released into the extracellular milieu (1), be embedded into the membrane via a lipid anchor (2) or a C-terminal transmembrane segment (3). In silico data also suggest that Sec substrates may be anchored to the cell wall in an archaeosortase-dependent manner (4) and a number of type IV pilin-like proteins have been shown to assemble into cell appendages (5). In I. hospitalis, an additional outer membrane (OM) is present and periplasmic vesicles (6) are thought to play a role in the trafficking of secreted and outer membrane proteins across the periplasmic space. See text for details. Cell components are not drawn to scale.

Figure 2

Detection of extracellular protein activities. (A) Overlay assay with colonies of S. acidocaldarius wt (right) and ΔsulAB mutant (left) grown on a lawn of S. solfataricus strain P2. Clear halos surrounding the colonies indicate killing of archaea by the sulfolobicins. Image reproduced with permission from Ellen et al. (2011). (B) Iodine vapor staining of H. volcanii expressing wild-type (left) or signal sequence mutated (right) α-amylase grown on rich medium supplemented with 0.2% soluble starch. Clear halos surrounding colonies indicate starch hydrolysis by extracytoplasmic α-amylase (Rose et al., 2002).

Figure 3

Archaeal surface structures. (A) Tetrathionate hydrolase from A. hospitalis YS8 assembles into zipper-like particles on the cell surface. Image reproduced with permission from Krupovic et al. (2012). (B) Electron micrographs of M. maripaludis cells expressing type IV pilus-like structures. Arrows indicate EppA-processed pili, while additional, thicker structures, are PibD-processed flagella. Samples were negatively stained with 2% phosphotungstic acid. Image reproduced with permission from (VanDyke et al., 2008). (C) Non-type IV Mth60 fimbriae of planktonic M. thermoautotrophicus cells by staining with AlexaFluor^®488 (Thoma et al., 2008). Image courtesy of R. Wirth, University of Regensburg, Germany. (D) Secreted halomucin complexes (stained green with specific antibody coupled to fluorescein) surrounds quadratic Haloarcula marismortui cell (stained red by Nile blue for polyhydroxy butyrate). Unpublished image, courtesy of D. Oesterhelt, Max Planck Institute of Biochemistry, Martinsried, Germany.

Figure 4

Ignicoccus hospitalis protein transport into and across the outer membrane. (A) Localization of A₁A_O ATP synthase on I. hospitalis outer membrane by EM of ultrathin sections and labeled with antibodies specifically raised against the purified 440-kDa ATPase complex (Kuper et al., 2010). Image courtesy of T. Heimerl, H. Huber, and R. Rachel, University of Regensburg, Germany. (B) Electron micrographs of I. hospitalis ultrathin sections. Outer membrane of I. hospitalis and the cell surface of N. equitans in direct contact (Junglas et al., 2008). Image courtesy of T. Heimerl, H. Huber, and R. Rachel, and image (B) courtesy of H. Huber and R. Rachel, University of Regensburg, Germany.