Fig. 5: HDLBP promotes ER translation and synthesis of secretory and transmembrane proteins. | Nature Communications

Fig. 5: HDLBP promotes ER translation and synthesis of secretory and transmembrane proteins.

From: HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins

Fig. 5: HDLBP promotes ER translation and synthesis of secretory and transmembrane proteins.The alternative text for this image may have been generated using AI.

a Schematic overview of the ribosome profiling experiment in HEK293 parental and HDLBP KO cells. Western analysis shows the absence of HDLBP in KO cells. b Membrane-bound HDLBP target mRNAs were split into three similarly sized groups based on their cross-linking signal in the CDS (indicated in parentheses). Differences in translation efficiency (HDLBP KO vs. WT) were compared between groups. A two-sided Wilcoxon rank-sum test was used to test for significance. c pSILAC analysis of newly synthesized proteins in HEK293 parental and HDLBP KO cells. SILAC heavy vs. medium ratios (H/M) reflect changes in protein synthesis upon HDLBP KO and were quantified in membrane fractions. Proteins were split into three similarly sized groups based on their cross-linking signal in the CDS (indicated in parentheses). SILAC ratios were compared between groups using a two-sided Wilcoxon rank-sum test. d Proteins were split into three similarly sized groups based on their PAR-CLIP signal and membrane localization (indicated in parentheses). SILAC ratios were compared between groups using a two-sided Wilcoxon rank-sum test. e Parental and HDLBP KO cells were transfected with secreted Gaussia luciferase (Gluc) construct. Gluc activity was quantified in the medium and normalized to the intracellular Firefly luciferase (Fluc) activity. Each of the four replicate experiments (r1–r4) was carried out with five technical replicates. Data were presented as mean values ± SD. f Parental and HDLBP KO cells were transfected secreted alkaline phosphatase (SEAP) construct. SEAP signal was quantified in medium and normalized to the intracellular Firefly luciferase (Fluc) activity. Each experiment was carried out in five technical replicates. Data were presented as mean values ± SD. g Western analysis of A549 cells stably transfected with construct overexpressing HDLBP (OE). h SEAP activity, relative to Fluc, was measured in HEK293 and A549 cells with different HDLBP protein levels (KO knockout, WT wild-type, HDLBP/OE overexpression). The experiment was carried out five times with at least five technical replicates. Data were presented as mean values ± SD. i Ribosome P-site coverage around targeting signals (signal peptides and transmembrane helices) was compared between HEK293 HDLBP knockout (KO) vs. parental (WT) cells. P-site coverage was scaled to the coverage in codons 20–40 of each mRNA. A rolling mean of 5 nt was used to smooth the profiles. Absolute numbers of analyzed mRNAs are given, median signal peptide and first transmembrane helix lengths are indicated with a vertical dotted line. a–i Source data are provided as a Source Data file.

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