Protein SortingII.pptx

Protein Sorting II Molecular Biology Overview Protein Sorting Protein Sorting Signals Mitochondria Sorting A specific sequence in mitochondrial proteins is recognized by import receptor that bring the protein to translocator to undergo posttranslational translocation In mitochondria this process is...

Protein Sorting II Molecular Biology Overview Protein Sorting Protein Sorting Signals Mitochondria Sorting A specific sequence in mitochondrial proteins is recognized by import receptor that bring the protein to translocator to undergo posttranslational translocation In mitochondria this process is even more complicated than in the ER as it requires translocation to both mitochondria membranes Peroxisome Proteins with peroxisome signals are recognized by PEX5 receptor PEX5 will transport protein to a receptor in the surface of peroxisome to be translocated inside peroxisome Therefore, there is NO cotranslational translocation: proteins are already translated when are translocated Proteins are translocated unfolded and properly fold once inside the peroxisome Nuclear Transport One the most important trafficking that exist in cells. Proteins involved in DNA metabolims like Histones, transcription factors, polymerases, helicases, primases… are made in the cytoplasm and then transported into the nucleus to perform its functions Some proteins constantly shuttle in and out of the nucleus while other are transported there remain in the nucleus Regulation of nuclear localization play an important regulatory for nuclear proteins. Nuclear membrane Made of two membranes, the inner (INM) and outer nuclear membrane (ONM) The ONM is in continuum with the ER Nuclear Pore Complex One of the biggest complexes on cells (16x bigger than ribosomes) Made out of multiple copies of over 30 different proteins It has an octagonal shape and is embedded between the IMN and ONM In the nucleoplasm NPC for a basket-like structure where is the cytoplasm side several proteins create filamentous structures Nuclear Pore Complex One of the biggest complexes on cells (16x bigger than ribosomes) Made out of multiple copies of over 30 different proteins It has an octagonal shape and is embedded between the IMN and ONM In the nucleoplasm NPC for a basket-like structure where is the cytoplasm side several proteins create filamentous structures How are protein targeted to the nucleus? Because they posses a Nuclear Localization Sequence (NLS) Proteins transport Importin a and Importin b recognize NLS in protein and direct them to the nuclear pore where they will translocated into the nucleus This is the only protein sorting mechanism where proteins are already folded by the time they get translocated. As any other process, it requires energy to be spend in the process NLS Although there are few variants , NLS tend to have a very basic short sequence. The first NLS discovered was part of a virus protein and it had the following sequence: – PKKKRKV Since then other similar sequence have been found and a K-K/R-X-K/R consensus sequence have been proposed Importins Importina and Importinb (also called Karyopherins) form a complex where importing recognizes a binds NLS in other protein and Importinb recognized the exterior filaments of the nuclear pore The Ran-GDP/GTP cycle is the key step in nuclear transport Because it provides the energy for transport and because it provides directionality The key is the gradient between RanGTP and RanGDP inside and outside the nucleus The Ran-GDP/GTP cycle is the key step in nuclear transport Because it provides the energy for transport and because it provides directionality The key is the gradient between RanGTP and RanGDP inside and outside the nucleus The Ran-GDP/GTP cycle is the key step in nuclear transport Because Ran-GEF remains in the nucleus and RanGAP in the cytoplasm, there is a much higher concentration of Ran-GTP in the nucleus and RanGDP in the cytoplasm Nuclear transport The second key element of this system is the fact than Ran-GTP has greater affinity for importina than RanGDP. Therefore in the nucleus importina tends to bind RaNGTP whereas in the cytoplasm is free to bind the cargo protein Nuclear transport The CargoImportin complex can freely pass the nuclear pore. The key then is that one it passes to the nucleus the cargo from importin because importing has great affinity for Ran-GTP which is only present in the nucleus Nuclear transport The Importin-RanGTP complex can then pass freely to the cytoplasm where Ran-GTP will be transformed into RanGDP. This causes the release of Ran protein from importing which now is free to bind a cargo protein and start the cycle again Every cycle consumes of GTP molecule Nuclear Export NLS containing proteins are actively transported inside the nucleus If such proteins need to be shuttling in and out the nucleus then it needs to have an mechanism to transported backwards from the nucleus into the cytoplasm. Proteins that shuttle in and out the nucleus have a Nucleus Export Sequence (NES) in addition to an NLS Nuclear export Proteins that shuttle in and out the nucleus posses a NES that is recognize by Exportin1 (Xpo1), who also binds RanGTP Once in the cytoplasm, RanGTP is transformed into RanGDP and the whole complex dissociate mRNA transport Remember that nuclear pore also serves to transport mRNA from the nucleus into the cytoplasm to be translated in the ribosomes. mRNA binds proteins that will aid in bringing mRNA to the nuclear pore and its subsequent translocation