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The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.

XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 angstrom cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.

Xbp1, a protein involved in the unfolded protein response, is a rare example of a mammalian protein that contains a well-defined translational arrest peptide (AP). In order to define the critical residues in the Xbp1u AP, and to search for variants with stronger arrest potency than the wildtype Xbp1u AP, we have carried out a full mutagenesis scan where each residue in the AP was replaced by the other 19 natural amino acids. We find that 10 of the 21 mutagenized positions are optimal already in the wildtype Xbp1 AP, while certain mutations in the remaining residues lead to a strong increase in the arrest potency. Xbp1 has thus evolved to induce an intermediate level of translational arrest, and versions with much stronger arrest efficiency exist. We further show Xbp1- induced translational arrest is reduced in response to increased tension in the nascent chain, making it possible to carry out studies in mammalian systems of cotranslational processes such as membrane protein assembly and protein folding by using suitable Xbp1 AP variants as “force sensors”, as has been done previously in E. coli using bacterial APs.