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Whole proteome of HeLa KTN1 KO rescued by a single variant of KTN1-FLAG

ABSTRACT: The endoplasmic reticulum (ER) forms sheet or tubule organization, and functions in regulating various cellular processes. Kinectin 1 (KTN1), an integral ER sheet protein involved in ER organization and translation elongation, has alternative exons on the cytoplasmic side, but their roles remain elusive. Here we found that the multi-tRNA synthetase complex (MSC), which is composed of eight aminoacyl-tRNA synthetases (ARSs) and three non-enzymatic proteins, specifically interacts with KTN1 depending on the alternative exon V2 in mouse and human cells. Through a combination of KTN1 knockout and rescue using variant-specific KTN1 expression, we show that the V2 exon is essential for MSC recruitment to the ER, where it contributes to the formation of rough ER stack organization. Our study thus identifies a novel role of KTN1 splicing variant in regulating the ER localization of the MSC, which is likely related to ex-translational activities of MSC to regulate ER sheet organization.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Keiko Nakayama

PROVIDER: PXD062552 | JPOST Repository | Thu Oct 30 00:00:00 GMT 2025

REPOSITORIES: jPOST

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			Action	DRS
	052724%20samples_1_4_5_8_fixed%20report.pg_matrix.xlsx	Xlsx
	20240509_EVO60_FMS45_12uDIA_Hosogane_sample01_rep01.raw	Raw
	20240509_EVO60_FMS45_12uDIA_Hosogane_sample01_rep02.raw	Raw
	20240509_EVO60_FMS45_12uDIA_Hosogane_sample01_rep03.raw	Raw
	20240509_EVO60_FMS45_12uDIA_Hosogane_sample01_rep04.raw	Raw

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KTN-FLAG-IP for identification of KTN1-variant specific binding proteins

Project description:The endoplasmic reticulum (ER) forms sheet or tubule organization, and functions in regulating various cellular processes. Kinectin 1 (KTN1), an integral ER sheet protein involved in ER organization and translation elongation, has alternative exons on the cytoplasmic side, but their roles remain elusive. Here we found that the multi-tRNA synthetase complex (MSC), which is composed of eight aminoacyl-tRNA synthetases (ARSs) and three non-enzymatic proteins, specifically interacts with KTN1 depending on the alternative exon V2 in mouse and human cells. Through a combination of KTN1 knockout and rescue using variant-specific KTN1 expression, we show that the V2 exon is essential for MSC recruitment to the ER, where it contributes to the formation of rough ER stack organization. Our study thus identifies a novel role of KTN1 splicing variant in regulating the ER localization of the MSC, which is likely related to ex-translational activities of MSC to regulate ER sheet organization.

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Project description:Kinectin 1 (KTN1) is an integral endoplasmic reticulum (ER) sheet protein that functions in ER organization and translation elongation. Alternative splicing introduces sequence diversity into the cytoplasmic region of KTN1, but the functional relevance of these variants has remained unclear. Here we show that the multi–tRNA synthetase complex (MSC), composed of eight aminoacyl-tRNA synthetases and three nonenzymatic proteins, specifically interacts with KTN1 in a manner dependent on the alternative exon V2 and glutamine-tRNA synthetase (QARS). Using V2 exon-specific knockout cells and KTN1 knockout cells with variant-specific resucue, we found that the V2 exon is required for KTN1-mediated recruitment of the MSC to the ER, where it promotes the formation of rough ER stacks. These findings define a variant-specific role for KTN1 in anchoring the MSC to the ER, and suggest that this interaction underlies a noncanonical activity of the MSC in regulating ER sheet organization.

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