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Affinity-purification MS of Drosophila melanogaster’s dSetDB1

ABSTRACT: Evaluation of binding partners of wild-type endogenously tagged flag-HA-dSetDB1 versus catalytic dead endogenously tagged flag-HA-250 dSetDB1 in Drosophila melanogaster early embryos

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Embryo

SUBMITTER: Alejandro Gomez-Auli

LAB HEAD: Gerhard Mittler

PROVIDER: PXD045480 | Pride | 2024-06-07

REPOSITORIES: pride

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			Action	DRS
	00000952___T180_rep1_wt_inj01.raw	Raw
	00000952___T180_rep1_wt_inj02.raw	Raw
	00000953___T180_rep2_wt_inj01.raw	Raw
	00000953___T180_rep2_wt_inj02.raw	Raw
	00000954___T180_rep3_wt_inj01.raw	Raw

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A novel C-terminal DxRSDxE motif in ceramide synthases involved in dimer formation

Project description:Ceramide is synthesized via the ceramide synthases (CerSs), six of which have been identified in mammalian cells, with each using a unique subset of acyl-CoAs for ceramide synthesis. The CerSs are part of a larger gene family, the Tram-Lag-CLN8 (TLC) domain family. We now identify a unique, C-terminal motif, the DxRSDxE motif, which is only found in CerS and not in other TLC family members. Deletion of this motif in either CerS2-HA or in CerS6-Flag did not affect the ability of either to generate ceramide using both an in vitro assay and upon metabolic labeling, but deletion of this motif did affect the activity of CerS2-HA when co-expressed with CerS6-Flag. Surprisingly, transfection of cells with either CerS2-HA or CerS6-Flag lacking the motif did not result in changes in cellular ceramide levels. CerS2-HA and CerS6-Flag interact with each other, as shown by immunoprecipitation, but deletion of the DxRSDxE motif impedes this interaction. Moreover, proteomics analysis of cells transfected with CerS6Δ338-344-Flag indicated that deletion of the C-terminal motif impacted cellular protein expression, and in particular, levels of ORMDL1, a negative regulator of sphingolipid synthesis. We suggest that this novel C-terminus motif regulates CerS dimer formation and thereby impacts ceramide synthesis.

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Convergence of distinct RNA-silencing pathways on GW182/Tnrc6 [RIP-seq]

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