Intercompatibility of eukaryotic and Asgard archaea ribosome-translocon machineries

In all domains of life, the ribosome-translocon complex inserts nascent transmembrane proteins into, and processes and transports signal peptide-containing proteins across, membranes. Eukaryotic translocons are anchored in the endoplasmic reticulum, while the prokaryotic complexes reside in cell membranes. Phylogenetic analyses indicate the inheritance of eukaryotic Sec61/oligosaccharyltransferase/translocon-associated protein translocon subunits from an Asgard archaea ancestor. However, the mechanism for translocon migration from a peripheral membrane to an internal cellular compartment (the proto-endoplasmic reticulum) during eukaryogenesis is unknown. Here we show compatibility between the eukaryotic ribosome-translocon complex and Asgard signal peptides and transmembrane proteins. We find that Asgard translocon proteins from Candidatus Prometheoarchaeum syntrophicum strain Candidatus Prometheoarchaeum syntrophicum strain MK-D1, a Lokiarchaeon confirmed to contain no internal cellular membranes, are targeted to the eukaryotic endoplasmic reticulum on ectopic expression. Furthermore, we show that the cytoplasmic domain of Candidatus Prometheoarchaeum syntrophicum strain MK-D1 oligosaccharyltransferase 1 (ribophorin I) can interact with eukaryotic ribosomes. Our data indicate that the location of existing ribosome-translocon complexes, at the protein level, determines the future placement of yet-to-be-translated translocon subunits. This principle predicts that during eukaryogenesis, under positive selection pressure, the relocation of a few translocon complexes to the proto-endoplasmic reticulum will have contributed to propagating the new translocon location, leading to their loss from the cell membrane.


Fig. S1 :
Fig. S1: Pearson correlation evaluation of the colocalization of EGFP-tagged proteins with the mCherry-ER marker from Fig. 1.A, S-layer, B, OST1 and C, TRAPa EGFP-tagged MK-D1 proteins show positive correlation (R = 0.69-0.70)indicating that these proteins associate with the ER.D, EGFP shows correlation close to zero (R = 0.09) indicating no directed association with the ER.Scale bar = 20 µm.Scale bar = 2.3 µm in the magnified images.

Fig. S2 :
Fig. S2: Colocalization of EGFP-tagged proteins with the mCherry plasma membrane marker as a control for Fig. 1.A, S-layer, B, OST1 and C, TRAPa MK-D1 EGFP-tagged proteins fluorescence (EGFP, green) fall inside the plasma membrane marker (mCherry-PM, magenta)indicating that these proteins are not associated with the plasma membrane.Scale bar = 20 μm.

Fig. S5 :
Fig. S5: AF2 statistics for the structure predictions in Fig. 2. AlphaFold2 co-predictions of human Sec61α with the signal peptides of A, MK-D1 S-layer, B, MK-D1 OST1, C, Human OST1, D, MK-D1 TRAPα, E, MK-D1 TRAPβ.The intermolecular interactions with the signal peptides are highlighted by blue arrows in the PAE plots.

Fig. S6 :
Fig. S6: The entire Western blot for the data shown in Fig. 3.The Western blot was produced from total cell samples probed with an anti-EGFP primary antibody.+ and -indicate the cells were grown in the presence or absence of tunicamycin, an N-linked glycosylation inhibitor, respectively.gEGFP refers to EGFP with an N-glycosylation acceptor site but without a signal peptide.This construct is not targeted to the ER, and its migration position (size, black arrow) is equivalent to the processed, non-glycosylated signal peptide-EGFP chimeras.Migration at higher molecular weight positions, relative to gEGFP in the + tunicamycin lanes indicate lack of cleavage of the signal peptides.Migration at higher molecular weight positions of each chimera in thetunicamycin lane, relative to the + tunicamycin lane, indicates glycosylation.The full Western blot is shown in Fig. S6.MW, molecular weight markers labelled in kDa.

Fig. S9 :
Fig. S9: AF2 statistics for the structure predictions for the segments used to build the MK-D1 translocon complex in Fig. 7A.

Fig. S12 :
Fig. S12: Potential ribosome binding residues on the OST1 cytosolic domain.A, Rainbowcolored cartoons of the MK-D1 OST1 cytosolic domain superimposed onto the human OST1 cytosolic domain structure (B, PDB 8B6L).Selected basic residues are shown and labeled.C,D Surface charge representations of A and B, respectively.A portion of the ribosome is shown with RNA in gray and proteins in cyan.E, A structure-based sequence alignment of MK-D1 and human OST1 cytosolic domains.The selected basic residues in A,B are highlighted in boxes.The residues were selected based on their proximity to the ribosome in the model (A) or structure (B).