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Isolation and MS characterisation of recombinant human septin octameric complexes containing distinct SEPT9 isoforms

ABSTRACT: Native human septins form heterooctameric complexes containing different SEPT9 isoforms. To enable studies of SEPT9 function, we established a protocol for the isolation of recombinant human octamers harboring distinct SEPT9 isoforms. Mass spectrometry analysis is used to assess the purity, integrity and stoichiometry of septins in the purified complex.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Luc Camoin

LAB HEAD: Marseille Proteomics

PROVIDER: PXD023857 | Pride | 2021-07-27

REPOSITORIES: Pride

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			Action	DRS
	A.raw	Raw
	A_20200813134739.raw	Raw
	A_2Runs.msf	Msf
	C.raw	Raw
	C_20200813210248.raw	Raw

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Project description:Septins are a family of multimeric GTP-binding proteins, which are abnormally expressed in cancer. Septin 9 (SEPT9) is an essential and ubiquitously expressed septin with multiple isoforms, which have differential expression patterns and effects in breast cancer cells. It is unknown, however, if SEPT9 isoforms associate with different molecular networks and functions. Here, we performed a proteomic screen in MCF-7 breast cancer cells to identify the interactome of GFP-SEPT9 isoforms 1, 4 and 5, which vary in the length of their N-terminus. While all three isoforms associated with SEPT2 and SEPT7, the truncated SEPT9_i4 and SEPT9_i5 interacted with septins of the SEPT6 group more promiscuously than SEPT9_i1, which bound predominately SEPT8. Spatial mapping and functional clustering of non-septin partners showed isoform-specific differences in interactions with proteins of distinct subcellular organelles (nuclei, centrosomes, cilia) and functions (signaling, degradation). Notably, the interactome of the full length SEPT9_i1 was more enriched in cytoskeletal regulators, while the truncated SEPT9_i4 and SEPT9_i5 exhibited preferential and isoform-specific interactions with nuclear and signaling factors as well as ubiquitinating enzymes. These data provide evidence for isoform-specific interactions, which arise from truncations in the N-terminal extensions of SEPT9, and point to novel roles in the pathogenesis of breast cancer.

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