ArtA-Dependent Processing of a Tat Substrate Containing a Conserved Tripartite Structure That Is Not Localized at the C Terminus

Abstract

Most prokaryote-secreted proteins are transported to the cell surface using either the general secretion (Sec) or twin-arginine translocation (Tat) pathway. A majority of secreted proteins are anchored to the cell surface, while the remainder are released into the extracellular environment. The anchored surface proteins play a variety of important roles in cellular processes, ranging from facilitating interactions between cells to maintaining cell stability. The extensively studied S-layer glycoprotein (SLG) of , previously thought to be anchored via C-terminal intercalation into the membrane, was recently shown to be lipidated and to have its C-terminal segment removed in processes dependent upon archaeosortase A (ArtA), a recently discovered enzyme. While SLG is a Sec substrate, analyses presented here reveal that, of eight additional ArtA substrates predicted, two substrates also contain predicted Tat signal peptides, including Hvo_0405, which has a highly conserved tripartite structure that lies closer to the center of the protein than to its C terminus, unlike other predicted ArtA substrates identified to date. We demonstrate that, even given its atypical location, this tripartite structure, which likely resulted from the fusion of genes encoding an ArtA substrate and a cytoplasmic protein, is processed in an ArtA-dependent manner. Using an Hvo_0405 mutant lacking the conserved "twin" arginines of the predicted Tat signal peptide, we show that Hvo_0405 is indeed a Tat substrate and that ArtA substrates include both Sec and Tat substrates. Finally, we confirmed the Tat-dependent localization and signal peptidase I (SPase I) cleavage site of Hvo_0405 using mass spectrometry. The specific mechanisms that facilitate protein anchoring to the archaeal cell surface remain poorly understood. Here, we have shown that the proteins bound to the cell surface of the model archaeon , through a recently discovered novel ArtA-dependent anchoring mechanism, are more structurally diverse than was previously known. Specifically, our results demonstrate that both Tat and Sec substrates, which contain the conserved tripartite structure of predicted ArtA substrates, can be processed in an ArtA-dependent manner and that the tripartite structure need not lie near the C terminus for this processing to occur. These data improve our understanding of archaeal cell biology and are invaluable for subcellular localization predictions of archaeal and bacterial proteins.

Conserved residues are critical for Haloferax volcanii archaeosortase catalytic activity: Implications for convergent evolution of the catalytic mechanisms of non‐homologous sortases from archaea and bacteria

“…We have recently been able to directly demonstrate ArtA‐dependent processing of a PGF tripartite structure containing protein, Hvo_0405. As the PGF tripartite structure of this H. volcanii ArtA substrate is located toward the center of the protein, rather than near the C‐terminus, it is an ideal reporter protein for studies on ArtA processing since the processed N‐ and C‐terminal proteins derived from Hvo_0405 can be readily observed on an LDS‐PAGE gel (Abdul Halim et al ., ). While the conditions under which chromosomal hvo_0405 is expressed are not yet known, to demonstrate ArtA‐dependent processing of this protein, we have expressed His‐tagged Hvo_0405 in trans under the control of the tryptophanase promoter (p. tnaA ) from the plasmid pTA963, and used antibodies against the N‐terminus of Hvo_0405His and the C‐terminal His‐tag to detect the processed proteins in immunoblot analyses.…”

“…While the conditions under which chromosomal hvo_0405 is expressed are not yet known, to demonstrate ArtA‐dependent processing of this protein, we have expressed His‐tagged Hvo_0405 in trans under the control of the tryptophanase promoter (p. tnaA ) from the plasmid pTA963, and used antibodies against the N‐terminus of Hvo_0405His and the C‐terminal His‐tag to detect the processed proteins in immunoblot analyses. Our results showed that Hvo_0405 can indeed be effectively used as a reporter substrate in studies of ArtA‐dependent processing (Abdul Halim et al ., ). Thus, we sought to determine whether the ArtA‐GFP replacement mutants can cleave this ArtA substrate.…”

“…For detection of the Hvo_0405 N‐terminus, the rabbit anti‐Hvo_0405‐N‐term serum (Abdul Halim et al ., ) was used at a 1:10,000 dilution in 3% BSA in PBS with sodium azide as the primary antibody. For the secondary antibody, the HRP‐conjugated Amersham ECL anti‐rabbit IgG from donkey (GE) was used at a 1:60,000 dilution in 10% nonfat milk in PBS.…”

“…In vivo studies of the Haloferax volcanii archaeosortase A (ArtA), confirmed that it is indeed required for processing of proteins containing the conserved C‐term motif, including a PGF‐motif followed by an H‐domain and positively charged residues (Abdul Halim et al ., , ). However, unlike sortase substrates, which are anchored to the peptidoglycan cell wall, ArtA substrates are covalently attached to a lipid embedded in the cytoplasmic membrane following C‐terminal processing (Abdul Halim et al ., ). Moreover, in contrast to sortase A substrates, which appear to be secreted exclusively via the general secretory (Sec) pathway, we recently determined that some ArtA substrates are translocated across the cytoplasmic membrane via the Twin Arginine Transport (Tat) pathway while others are translocated via the Sec pathway (Schneewind and Missiakas, ; Goosens and van Dijl, ; Abdul Halim et al ., ).…”

Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea

“…Recently, a novel mechanism was discovered whereby proteins are anchored to the membrane through a lipid moiety covalently attached to a processed C terminus. In archaeal cells, processing and lipid modification of these C-terminal anchored proteins are mediated by enzymes known as archaeosortases, with archaeosortase A (ArtA) of the model archaeon Haloferax volcanii being the most studied example (10)(11)(12)(13). Proteins recognized and processed by H. volcanii ArtA contain a distinct C-terminal tripartite structure consisting of a conserved PGF motif, followed by a hydrophobic domain and then a stretch of positively charged residues.…”