Health & Environmental Research Online (HERO)


Print Feedback Export to File
975894 
Journal Article 
Abstract 
Neuronal nitric oxide synthase trafficking into mitochondria 
Carreras, MC; Franco, MC; Molinas, MF; Gonzalez, AG; Finocchietto, PF; Converso, DP; Poderoso, JJ 
2008 
Yes 
Free Radical Biology and Medicine
ISSN: 0891-5849
EISSN: 1873-4596 
45 
Suppl. 
S111-S111 
English 
WOS:000260867900314 
is part of a larger document 3452652 SFRBM's 15th Annual Meeting: Program and Abstracts
Mitochondrial nitric oxide synthase (mtNOS) has been described as a post-translational modified neuronal NOS-alpha (130 kDa). Modulation of mtNOS was observed in different situations; however, the mechanism of translocation into mitochondria is still unknown. We therefore studied a) Rat liver and brain mitochondrial translocation of nNOS-alpha in experimental hypothyroidism in vivo and b) HEK 293 cells transfection with two vectors: wild type nNOS and delta-pdz nNOS. the processing of nNOS protein was assayed in Western blots by using antibodies (Abs) against different domains (aa1-100, 144-262, 1095-1289 and 1380-1429 or against the V5 tag), and internalization of the protein by proteinase K to eliminate the protein attached to the cytosolic side of mitochondrial outer membrane. After 14 days of hypothyroidism, a pike of mitochondrial translocated nNOS expression and activity was detected in liver and brain. Apparent MW of translocated nNOS resulted reduced from 160 to 130 kDa. Since imported nNOS was not detected by antibodies against aa 1-100 but by all the other Abs, we confirmed that the 130 kDa mtNOS band looses the N-terminal PDZ domain during nNOS import. Wild type 160 kDa nNOS transfected to HEK 293 cells, that do not express either NOS isoform, was inserted in mitochondrial outer membrane and PK treatment cut the amino and carboxi terminal domains, resulting in a 130 kDa fragment only detected by Abs against aa144-262 and aa 1095-1289. Similar results were obtained with the 130 kDa delta–pdz nNOS, detecting a 120 kDa band after PK treatment. All the proteins were functional. These data suggest that nNOS is internalized in vivo loosing the PDZ domain and passes through the mitochondrial outer membrane translocation channels as a hairpin loop leading the extremes in the cytosolic side and susceptible to endogenous proteases or to PK treatment. c) Mitochondria from HEK 293 cells challenged with nNOS newly synthesized by a reticulocyte lysate translation system (TTS). in the in vitro assay, mitochondria from HEK 293 cells were incubated with nNOS newly synthesized by TTS and an aliquot was treated with PK resulting in a 130 kDa band still functional protein as in the transfection studies. 
Society for Free Radical Biology and Medicine 15th Annual Meeting 
Indianapolis, IN 
November 19-23, 2008