The ER anchoring and abundance of the EEF1B complex is affected by tissue-specific alternative EEF1D splicing.

The EEF1B complex plays a central role in translation elongation by reactivating EEF1A for the 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 physiological functions remain poorly characterized. In this study, we show that short EEF1D isoforms containing exon 5 interact with the ER-resident scaffold protein KTN1 and RRBP1, thereby anchoring the EEF1B complex to the ER. Mass spectrometry analyses 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 KO mice, this altered localization was accompanied by a reduction in EEF1B subunit abundance in multiple tissues, including the liver, although global protein synthesis rates remained unchanged. Together, these findings uncover an ER-anchoring mechanism controlled by alternative splicing that shapes the spatial organization and abundance of the elongation machinery in vivo.