Chloroplasts and mitochondria contain a family of putative preprotein and amino acid transporters designated PRAT. Here, we analyzed the role of two previously characterized PRAT protein family members, encoded by At3...Chloroplasts and mitochondria contain a family of putative preprotein and amino acid transporters designated PRAT. Here, we analyzed the role of two previously characterized PRAT protein family members, encoded by At3g49560 （HP30） and At5g24650 （HP30-2）, in planta using a combination of genetic, cell biological and biochemical approaches. Expression studies and green fluorescent protein tagging identified HP30-2 both in chloroplasts and mitochondria, whereas HP30 was located exclusively in chloroplasts. Biochemical evidence was obtained for an association of mitochondrial HP30-2 with two distinct protein complexes, one containing the inner membrane translocase TIM22 and the other containing an alternative NAD（P）H dehydrogenase subunit （NDCI） implicated in a respiratory complex 1-1ike electron trans- port chain. Through its association with TIM22, HP30-2 is involved in the uptake of carrier proteins and other, hydrophobic membrane proteins lacking cleavable N H2-terminal presequences, whereas HP30-2＇s interaction with NDC1 may permit controlling mitochondrial biogenesis and activity.展开更多
AIM:To develop a sensitive assay for screening compounds against hepatitis C virus (HCV).METHODS:The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling prote...AIM:To develop a sensitive assay for screening compounds against hepatitis C virus (HCV).METHODS:The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP),which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence.Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy.Cellular localization and protein levels were examined by Western blotting.RESULTS:HCV NS3/4A protease cleaved eYFP-MAVSfrom mitochondria to block the activation of interferon (IFN)-β promoter,thus resulting in downregulation of SEAP activity.The decrease in SEAP activity was proportional to the dose of active NS3/4A protease.Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A.CONCLUSION:Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors.This system will constitute a new tool to allow the efficient screening of HCV inhibitors.展开更多
基金supported by the Chaire d’Excellence Program of the French Ministry of National Education and Research(to CR)
文摘Chloroplasts and mitochondria contain a family of putative preprotein and amino acid transporters designated PRAT. Here, we analyzed the role of two previously characterized PRAT protein family members, encoded by At3g49560 （HP30） and At5g24650 （HP30-2）, in planta using a combination of genetic, cell biological and biochemical approaches. Expression studies and green fluorescent protein tagging identified HP30-2 both in chloroplasts and mitochondria, whereas HP30 was located exclusively in chloroplasts. Biochemical evidence was obtained for an association of mitochondrial HP30-2 with two distinct protein complexes, one containing the inner membrane translocase TIM22 and the other containing an alternative NAD（P）H dehydrogenase subunit （NDCI） implicated in a respiratory complex 1-1ike electron trans- port chain. Through its association with TIM22, HP30-2 is involved in the uptake of carrier proteins and other, hydrophobic membrane proteins lacking cleavable N H2-terminal presequences, whereas HP30-2＇s interaction with NDC1 may permit controlling mitochondrial biogenesis and activity.
基金Supported by The Natural Science Foundation of China,No.30600330,No.30671842,No.30672488,No.30700475,No.30771919and No.30700757the National High Technology Research and Development Program of China,No.2008AA02Z132+1 种基金Beijing Municipal Natural Science Foundation,No.5082016Mega-projects of Science Research for the 11th Five-Year Plan,No.2009ZX10004-4001
文摘AIM:To develop a sensitive assay for screening compounds against hepatitis C virus (HCV).METHODS:The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP),which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence.Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy.Cellular localization and protein levels were examined by Western blotting.RESULTS:HCV NS3/4A protease cleaved eYFP-MAVSfrom mitochondria to block the activation of interferon (IFN)-β promoter,thus resulting in downregulation of SEAP activity.The decrease in SEAP activity was proportional to the dose of active NS3/4A protease.Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A.CONCLUSION:Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors.This system will constitute a new tool to allow the efficient screening of HCV inhibitors.
