9.B.297.  The Archaeosortase/Exosortase/Rhomosortase/Cyanosortase (Sortase) Family 

Integral membrane archaeosortases recognize and remove carboxyl-terminal protein targetting signals about 25 amino acids long from secreted proteins (Haft et al. 2012). A genome that encodes one archaeosortase may encode over fifty target proteins. The best characterized archaeosortase target is the Halofax volcanii S-layer glycoprotein, an extensively modified protein with O- and N-linked glycosylations as well as a large prenyl-derived lipid modifications toward the C-terminus (Abdul Halim et al. 2013). Knockout of the archaeosortase A (artA) gene, or permutation of the motif Pro-Gly-Phe (PGF) to Pro-Phe-Gly in the S-layer glycoprotein, blocks attachment of the lipid moiety as well as blocking removal of the PGF-CTERM protein-sorting domain (Abdul Halim et al. 2015). Thus archaeosortase appears to be a transpeptidase like sortase, rather than a simple protease. 

Archaeosortases are related to exosortases, their uncharacterized counterparts in Gram-negative bacteria. The names of both families of proteins reflect roles analogous to sortases in Gram-positive bacteria, with which they lack significant sequence similarity. The sequences of archaeosortases and exosortases consists mostly of hydrophobic transmembrane helices, which sortases lack (Abdul Halim et al. 2018). Archaeosortases fall into a number of distinct subtypes, each responsible for recognizing sorting signals with a different signature motif. Archaeosortase A (ArtA) recognizes the PGF-CTERM signal, ArtB recognizes VPXXXP-CTERM, ArtC recognizes PEF-CTERM, and so on; one archaeal genome may encode two different archaeosortase systems (Giménez et al. 2015). 

In silico discovery of the myxosortases that process MYXO-CTERM and three novel prokaryotic C-terminal protein-sorting signals that share invariant Cys residues has been described (Haft 2024).  The LPXTG protein-sorting signal, found in surface proteins of various Gram-positive pathogens, was the founding member of a growing panel of prokaryotic small C-terminal sorting domains. Sortase A cleaves LPXTG, exosortases (XrtA and XrtB) cleave the PEP-CTERM sorting signal, archaeosortase A cleaves PGF-CTERM, and rhombosortase cleaves GlyGly-CTERM domains. Four sorting signal domains without previously known processing proteases are the MYXO-CTERM, JDVT-CTERM, Synerg-CTERM, and CGP-CTERM domains. These exhibit the standard tripartite architecture of a short signature motif, a hydrophobic transmembrane segment, and an Arg-rich cluster. Each has an invariant cysteine in its signature motif. Computational evidence strongly suggests that each of these four Cys-containing sorting signals is processed, at least in part, by a cognate family of glutamic-type intramembrane endopeptidases related to the eukaryotic type II CAAX-processing protease Rce1. For the MYXO-CTERM sorting signals of different lineages, their sorting enzymes, called myxosortases, include MrtX (MXAN_2755 in Myxococcus xanthus), MrtC, and MrtP, all with radically different N-terminal domains but with a conserved core. Related predicted sorting enzymes were also identified for JDVT-CTERM (MrtJ), Synerg-CTERM (MrtS), and CGP-CTERM (MrtA). This work establishes a major new family of protein-sorting housekeeping endopeptidases contributing to the surface attachment of proteins in prokaryotes. Homologs of the eukaryotic type II CAAX-box protease Rce1, a membrane-embedded endopeptidase found in yeast and human ER and involved in sorting proteins to their proper cellular locations, are abundant in prokaryotes. The bioinformatics paper by Haft 2024 identified several subgroups of the family as cognate endopeptidases for four protein-sorting signals processed by a previously unknown machinery. Sorting signals with newly identified processing enzymes include three novel ones, but also MYXO-CTERM, which had been the focus of previous experimental work in the model fruiting and gliding bacterium Myxococcus xanthus. These findings will substantially improve our understanding of Cys-containing C-terminal protein-sorting signals and of protein trafficking generally in bacteria and archaea. Some of these sortases are listed under TC subfamily 9.B.2.14 because they are more similar in sequence to members of 9.B.2.1 than to members of family 9.B.297.