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Thousands of human small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been annotated. Many alt-ORFs are co-encoded with canonical proteins in multicistronic configurations, but few of their functions are known. Here, we report the detection of alt-RPL36, a protein co-encoded with human RPL36. Alt-RPL36 partially localizes to the endoplasmic reticulum, where it interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 increases plasma membrane PI(4,5)P 2 levels, upregulates PI3K-AKT-mTOR signaling, and increases cell size. Alt-RPL36 contains four phosphoserine residues, point mutations of which abolish interaction with TMEM24 and, consequently, alt-RPL36 effects on PI3K signaling and cell size. These results implicate alt-RPL36 as an upstream regulator of PI3K-AKT-mTOR signaling. More broadly, the RPL36 transcript encodes two sequence-independent polypeptides that co-regulate translation via different molecular mechanisms, expanding our knowledge of multicistronic human gene functions.

Epidermal growth factor receptor (EGFR) activation by growth factors (GFs) relies on dimerization and allosteric activation of its intrinsic kinase activity, resulting in trans-phosphorylation of tyrosines on its C-terminal tail. While structural and biochemical studies identified this EGF-induced allosteric activation, imaging collective EGFR activation in cells and molecular dynamics simulations pointed at additional catalytic EGFR activation mechanisms. To gain more insight into EGFR activation mechanisms in living cells, we develop a Förster resonance energy transfer (FRET)-based conformational EGFR indicator (CONEGI) using genetic code expansion that reports on conformational transitions in the EGFR activation loop. Comparing conformational transitions, self-association and auto-phosphorylation of CONEGI and its Y845F mutant reveals that Y 845 phosphorylation induces a catalytically active conformation in EGFR monomers. This conformational transition depends on EGFR kinase activity and auto-phosphorylation on its C-terminal tail, generating a looped causality that leads to autocatalytic amplification of EGFR phosphorylation at low EGF dose.

This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: probe incorporation mediated by enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches a picolyl azide (pAz) derivative to a 13-aa recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing pAz are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize pAz to perform PRIME labeling and to achieve CuAAC derivatization of pAz on live cells, fixed cells and purified proteins. Reagent preparations, including synthesis of pAz probes and expression of LplA, take 12 d, whereas the procedure for performing site-specific pAz ligation and CuAAC on cells or on purified proteins takes 40 min-3 h.