Fig 1.
P. blakesleeanus OCTIN crystals.
(A) The upper panel shows Phycomyces asexual fruiting body development tracked over the course of 10 hours. The stalk is a single-celled sporangiophore and the sphere at its tip contains nuclei that develop into asexual spores. The asterisk indicates the approximate region where protein crystals occur. The lower panel shows a close-up view of protein crystals within the sporangiophore central vacuole. (B) Three focal planes reveal the octahedral structure of a purified crystal. The bottom panel shows a cartoon of the crystal geometry. The lightest triangular face corresponds to the first panel. The darkest triangular face corresponds to the third panel. (C) The crystal-enriched fraction analyzed by SDS-PAGE. Two prominent proteins, p55 and p14 are indicated. The asterisk identifies a 46-kDa band whose peptides are mapped to the same region as p55 by mass spectrometry. (D) The cartoon depicts the full-length OCTIN sequence. Peptides identified from p14 and p55 are shown in blue and yellow, respectively. The N-termini of the mature proteins are indicated (arrowheads). The dashed line identifies the predicted region removed through proteolytic processing based on the molecular weight of p14. An asterisk marks the position of a stop-codon in the crystal-less mutant. (E) An organismal phylogeny showing the distribution of taxa where full-length OCTIN homologs are found. Names of these taxa are in colored or black labels. Gray colored groups do not contain OCTIN. OCTIN, octahedral crystal matrix protein.
Fig 2.
The OCTIN phylogeny indicates multiple HGT events.
The OCTIN ML phylogenetic tree supports bacteria-Mucorales, bacteria-Oomycetes, and bacteria-bacteria HGTs. Support values greater than 50 are shown as node labels. Values of 100 are represented by thick horizontal lines. The tree is rooted with a distant homolog, human FGE. Sequences with predicted N-terminal signal sequences, and gram-negative and gram-positive bacteria are marked with the indicated symbols. The various taxa are color-coded according to the legend. The full ML tree constructed from 127 OCTIN sequences is shown in S3A Fig. FGE, formylglycine-generating enzyme; HGT, horizontal gene transfer; ML, maximum likelihood; OCTIN, octahedral crystal matrix protein.
Fig 3.
The Phycomyces OCTIN crystal lattice is stabilized by intermolecular disulphide bonds.
(A) Conservation of cysteine residue position shown by sequence alignment. Positions exhibiting clade-specific conservation are shown in the color of the species to which they correspond. The positions of other cysteine residues are shown in black. Connected lines above the human FGE represent cysteine pairs that form disulphide bonds. The arrowhead indicates the catalytic cysteine pair. The FGE domain is indicated by the horizontal black bar. (B) Crystal-enriched fraction separated by SDS-PAGE in the presence (+) and absence (−) of the reducing agent 2-ME. P55, but not p14, migrates as a high-molecular–weight smear in the absence of 2-ME. Note that p46 also shifts in the absence of 2-ME, suggesting that it is a processing variant of p55. (C) Stills taken from a video recording the disassembly of Phycomyces OCTIN crystals by the reducing agent DTT (S1 Movie). (D) Synergistic disassembly of Phycomyces OCTIN crystals by SDS and DTT. While SDS alone is sufficient to completely shift p14 to the supernatant after centrifugation at 100,000 x g, only the combination of SDS and DTT has the same effect on p55. 2-ME, 2-Mercaptoethanol; DTT, dithiothreitol; FGE, formylglycine-generating enzyme; OCTIN, octahedral crystal matrix protein; P, pellet; S, supernatant; SDS, sodium dodecyl sulfate; T, total.
Fig 4.
Localization and assembly of bacterial and Phycomyces OCTIN upon ectopic expression.
(A) Terriglobus OCTIN assembles into patches in the periplasm. The Terriglobus OCTIN signal sequence fused to the N′-terminus of mCherry (SST-mCherry) is localized in a ring around the cell periphery, while a full-length OCTINT-mCherry fusion protein is localized in patches. (B) Western blotting shows the enrichment of SST-mCherry and OCTINT-HA in an isolated periplasmic fraction. Cytoplasmic mCherry serves as a control for contamination of the periplasmic fraction through cell lysis. (C) OCTINT-HA assembles into high-order oligomers. Periplasmic OCTINT-HA, but not SST-mCherry is pelleted by centrifugation at 100,000 x g. (D) OCTINT forms intermolecular disulphide bonds. OCTINT-HA migrates as a high-molecular–weight smear in the absence (−) but not presence (+) of 2-ME. (E) As with Phycomyces OCTIN crystals (Fig 3D), SDS and DTT synergize to promote the disassembly of OCTINT oligomers. (F) OCTINT-mCherry and SST-OCTINP-mCherry are targeted to the ER upon expression in mammalian cells. The ER is defined by sfGFP with an N-terminal SS and C′-terminal ER retention signal (KDEL). (G) Western blotting for OCTINT-HA and SST-OCTINP-HA expressed in mammalian cells shows that SST-OCTINP does not undergo proteolytic processing in the ER. Arrowhead indicates the expected position of p55. 2-ME, 2-Mercaptoethanole; BF, brightfield; DTT, dithiothreitol; ER, endoplasmic reticulum; FL; Fluorescence; OCTIN, octahedral crystal matrix protein; P, pellet; S, supernatant; SDS, sodium dodecyl sulfate; sfGFP, superfolder GFP, SS, signal sequence; T, total.