Residues in SRP9/14 essential for elongation arrest activity of the signal recognition particle define a positively charged functional domain on one side of the protein

The signal recognition particle (SRP) is a ubiquitous cytoplasmic ribonucleoprotein complex required for the cotranslational targeting of proteins to the endoplasmic reticulum (ER). In eukaryotes, SRP has to arrest the elongation of the nascent chains during targeting to ensure efficient translocation of the preprotein, and this function of SRP is dependent on SRP9/14. Here we present the results of a mutational study on the human protein h9/14 that identified and characterized regions and single residues essential for elongation arrest activity. Effects of the mutations were assessed both in cell-free translation/translocation assays and in cultured mammalian cells. We identified two patches of basic amino acid residues that are essential for activity, whereas the internal loop of SRP14 was found to be dispensable. One patch of important basic residues comprises the previously identified basic pentapetide KRDKK, which can be substituted by four lysines without loss of function. The other patch includes three lysines in the solvent-accessible α2 of h9. All essential residues are located in proximity in SRP9/14 and their basic character suggests that they serve as a positively charged platform for interactions with ribosomal RNA. In addition, they can all be lysines consistent with the hypothesis that they recognize their target(s) via electrostatic contacts, most likely with the phosphate backbone, as opposed to contacts with specific bases

Calreticulin mutations affect its chaperone function and perturb the glycoproteome

Calreticulin (CALR) is an endoplasmic reticulum (ER)-retained chaperone that assists glycoproteins in obtaining their structure. CALR mutations occur in patients with myeloproliferative neoplasms (MPNs), and the ER retention of CALR mutants (CALR MUT) is reduced due to a lacking KDEL sequence. Here, we investigate the impact of CALR mutations on protein structure and protein levels in MPNs by subjecting primary patient samples and CALR-mutated cell lines to limited proteolysis-coupled mass spectrometry (LiP-MS). Especially glycoproteins are differentially expressed and undergo profound structural alterations in granulocytes and cell lines with homozygous, but not with heterozygous, CALR mutations. Furthermore, homozygous CALR mutations and loss of CALR equally perturb glycoprotein integrity, suggesting that loss-of-function attributes of mutated CALR chaperones (CALR MUT) lead to glycoprotein maturation defects. Finally, by investigating the misfolding of the CALR glycoprotein client myeloperoxidase (MPO), we provide molecular proof of protein misfolding in the presence of homozygous CALR mutations.ISSN:2666-3864ISSN:2211-124