Protein targe ng to mitochondria chloroplasts and peroxisomes 1 2 3 Mitochondria and Chloroplasts Both surrounded by a double membrane a third internal membrane is also present in chloroplasts Each has its own DNA contained in a genome in the lumen inside the inner membrane Some proteins are made from own DNA on internal ribosomes and some are made in the cytoplasm from nuclear DNA and imported Both divide by ssion like bacteria Both can only be generated from pre exisDng mitochondria or chloroplasts but they to need import nuclear encoded proteins for proper funcDon Mitochondria and Chloroplasts Bacterial origins Maintenance and dynamics via ssion and fusion Mitochondrial structure and func on Surrounded by double membrane Outer membrane Inner membrane Infolded to create a large amount of internal membrane surface area Innermost space is called the matrix There is an intermembrane space Mitochondria produce ATP for cellular energy In Cytoplasm Glycolysis breaks down glucose to produce 2 ATP s and pyruvate In Mitochondria OxidaDve metabolism Citric Acid TCA Krebs cycle Pyruvate is transported into mitochondria and broken down to produce 30 more ATP Mitochondria are found throughout the cytoplasm of all eukaryoDc cells 1000 cell They are someDmes placed in speci c locaDons where energy need is greater They are oWen associated with microtubules and moved along microtubules by molecular motors Some cells have more because they have more energy needs muscle Mitochondria are generally cylindrical in shape but can fuse or elongate into tubes and change shape dynamic The role of changing morphology is not understood Chloroplasts Found throughout leaf cells along with mitochondria Surrounded by double membrane Internally have a third membrane layer a ened membranous stacks thylakoids Inside of sacs thylakoid space or lumen Outside of sacs stroma or matrix In the membrane of the thylakoids is the photosyntheDc machinery In the stroma is the carbohydrate synthesis machinery Use light energy to produce ATP then use ATP to make sugars glucose Mitochondria and chloroplasts both have their own genomes Mito DNA from several organisms have been completely sequenced Human 16 569 base pairs Large and small rRNA 22 transfer RNA s 13 di erent proteins Plant much larger and variable in size 90 of mitochondrial proteins are encoded by the nucleus So mitochondria has a mixture of proteins encoded by both nuclear and mitochondrial genes Some enzymes are mixtures of nuclear and mitochondrial subunits Mitochondria in di erent Dssues are slightly di erent Mitochondrial genomes in di erent species have overlapping sets of genes The ones that are not present in the mtDNA are in the nucleus It takes a certain set of genes to make a funcDoning mitochondria but whether those genes are nuclear or mitochondrial in origin varies with the species Chloroplast genome has been completely sequenced 121 024 bp 120 genes 4 rRNA s 20 ribosomal proteins various enzyme subunits Chloroplasts also have a mixture of proteins encoded by both nuclear and chloroplast genes Mitochondrial genome Chloroplast genome Mitochondria convert products of glycolysis into ATP Products of the citric acid cycle feed the electron transport chain establishing a proton gradient across the inner mitochondrial membrane used to power ATP synthase Chloroplasts use light energy to generate a proton gradient used to make ATP While the H gradients are set up across di erent compartments in mitochondria and chloroplasts they ul mately drive ATP synthesis in the matrix stroma of both Small Molecule Transport The outer membrane is like the outer membrane of bacteria it contains many types of Porins pore forming proteins which are barrel channels Small molecules and ions move freely between the intermembrane space and the cytoplasm The inner membrane has transporters that selecDvely move small molecules across the inner membrane into and out of the matrix ADP ATP exchanger uses H voltage gradient Pyruvate H cotransporter Phosphate H cotransporter ATP Synthase is a chimera of mitochondriaand nuclear encoded proteins ATP synthase is a molecular turbine Demonstra on of the rotary shaM ATP synthase anima on As the shaW rotates the subunits change shape causing ATP synthesis h p www youtube com watch v PjdPTY1wHdQ Targe ng proteins to various mitochondrial compartments 90 of mitochondrial proteins are nuclear encoded and made in cytoplasm How are these molecules targeted to the mitochondria and its various compartments Matrix Inner Membrane Outer membrane Intermembrane space Proteins encoded by the mitochondrial genome are made in mitochondrial matrix with internal ribosomes Targe ng proteins to mitochondria Nuclear encoded mitochondrial proteins have a targeDng signal Import is post transla onal transmembrane transport The rst step involves a targeDng signal direcDng proteins to the mitochondrial outer membrane This signal is an N terminal amphipathic helix with charged amino acids Necessary and su cient for import The signal is bound by a receptor protein in the mitochondrial outer membrane Mitochondrial genome encoded matrix proteins have no signal no need made in matrix Experimental paradigm for inves ga ng import of proteins into mitochondria Transport across the inner and outer membranes There is a receptor for the signal in the outer membrane Part of a special membrane pore complex TOM transporter outermembrane mitochondria This protein funcDons for all possible protein desDnaDons Outer membrane Inner membrane Matrix space Intermembrane space There are a number of inner membrane protein complexes that come into play depending upon the protein s ulDmate desDnaDon i e TIMs OXA etc Once exposed in the matrix the targeDng signal is cleaved o Transport of proteins into the matrix occurs across both membranes at once Protein is kept unfolded by cytosolic chaperones Signal sequence is recognized by receptor and TOM40 complex Protein is transported through TOM40 complex and TIM44 TIM23 17 complex at a contact site Matrix chaperone binds protein Matrix protease cleaves signal sequence Demonstra ng unfolded import and Tom Tim contact sites Three energy dependent steps in uptake of mitochondrial membrane proteins Targe ng to other loca ons involves secondary signal sequences varied Tom Tim complexes and associated membrane proteins Inner Membrane Targe ng Targe ng to other loca ons involves secondary signal sequences varied Tom Tim complexes and associated