The ER Anchoring and Abundance of the EEF1B Complex is Affected by Tissue Specific Alternative EEF1D Spicing

The EEF1B complex plays a central role in translation elongation by reactivating EEF1A for delivery of aminoacyl-tRNAs to the ribosome. Among its components, EEF1D undergoes alternative splicing to produce one long and several short isoforms, each with distinct N-terminal domains and tissue-specific expression patterns. Although the short isoforms are broadly expressed, their functional significance has remained unclear. In this study, we show that short EEF1D isoforms containing exon 5 interact with endoplasmic reticulum (ER)-resident scaffold protein KTN1 and RRBP1, thereby anchoring the EEF1B complex to the ER. Mass spectrometry of FLAG-tagged EEF1D identified these interactions, and deletion of exon 5 disrupted ER anchoring, resulting in diffuse cytoplasmic localization of the EEF1B complex. In exon 5 knockout mice, this altered localization was accompanied by reduced EEF1B subunit abundance in multiple tissues, including liver, although global protein synthesis rates remained unaffected. These findings uncover an ER-anchoring mechanism controlled by alternative splicing that shapes the spatial organization and abundance of the elongation machinery in vivo.