title: Targeting of pro-apoptotic TLR adaptor SARM to mitochondria: definition of the critical region and residues in the signal sequence.
abstract: The fifth and the most well-conserved member of the TLR (Toll-like receptor) adaptor, SARM (sterile α- and HEAT/armadillo-motif-containing protein), has been reported to be an important mediator of apoptosis. However, the exact cellular localization of SARM with respect to its role is unclear. In the present study we show that SARM specifically co-localizes with mitochondria. Endogenous SARM is mainly found in the mitochondria. We demonstrate that the N-terminal 27 amino acids (S27) of SARM, which is hydrophobic and polybasic, acts as a mitochondria-targeting signal sequence, associating SARM to the mitochondria. The S27 peptide has an inherent ability to bind to lipids and mitochondria. This sequence effectively translocates the soluble EGFP (enhanced green fluorescence protein) reporter into the mitochondria. Positioning S27 downstream of the EGFP abrogates its mitochondria-targeting ability. Transmission electron microscopy confirms the ability of S27 to import EGFP into the mitochondria. Importantly, by mutagenesis study, we delineated the specificity of the mitochondria-targeting ability to the arginine residue at the 14th position. The R14A SARM mutant also showed reduced apoptotic potential when compared with the wild-type. Taken together, S27, which is a bona fide signal sequence that targets SARM to the mitochondria, explains the pro-apoptotic activity of SARM.
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore.
authors.forename: Porkodi
authors.initials: P
authors.lastname: Panneerselvam
authors.forename: Laishram Pradeepkumar
authors.initials: LP
authors.lastname: Singh
authors.forename: Bow
authors.initials: B
authors.lastname: Ho
authors.forename: Jianzhu
authors.initials: J
authors.lastname: Chen
authors.forename: Jeak Ling
authors.initials: JL
authors.lastname: Ding
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D051186:Armadillo Domain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D016253:Microscopy, Immunoelectron; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051193:Toll-Like Receptors / Q000378:metabolism*
chemical_list: D051186:Armadillo Domain Proteins; D003598:Cytoskeletal Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C435689:SARM1 protein, human; C543961:SARM1 protein, mouse; D051193:Toll-Like Receptors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins