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FLAG-EEF1D-IP for identification of EEF1D-variant specific binding proteins

ABSTRACT: 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 exonâ€¯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.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Masaki Hosogane

PROVIDER: PXD075543 | JPOST Repository | Sat May 30 00:00:00 GMT+01:00 2026

REPOSITORIES: jPOST

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	20220608_IP-MS_DrHosogane_01.raw	Raw
	20220608_IP-MS_DrHosogane_02.raw	Raw
	20220608_IP-MS_DrHosogane_03.raw	Raw
	20220608_IP-MS_DrHosogane_04.raw	Raw
	20260109_DrHosogane_IP_MS_01.raw	Raw

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Project description: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 exonâ€¯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.

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