Editing Protein targeting (section)

=== Mitochondria ===
[[File:Overview of proteins targeted to the mitochondira.png|thumb|Overview of the major protein import pathways of mitochondria.]]
[[File:Carrier pathway for proteins to inner membrane.png|thumb|The carrier pathway for proteins targeted to the mitochondrial inner membrane.]]
While some proteins in the mitochondria originate from [[mitochondrial DNA]] within the organelle, most [[Mitochondrion|mitochondrial]] [[protein]]s are synthesized as [[cytosolic]] precursors containing uptake [[peptide signal]]s.<ref>{{Cite book |last=Cox M, Doudna J, O'Donnel M |title=Molecular Biology Principles and Practice |publisher=W.H. Freeman and Company |year=2015 |isbn=978-1-319-15413-4 |edition=2nd |location=New York, NY}}</ref><ref name="Araiso Y, Endo T-2022">{{Cite journal |last=Araiso Y, Endo T |date=2022 |title=Structural overview of the translocase of the mitochondrial outer membrane complex |journal=Biophys Physicobiol |volume=19 |pages=e190022 |doi=10.2142/biophysico.bppb-v19.0022 |pmid=35859989 |pmc=9260164 }}</ref><ref name="Eaglesfield R, Tokatlidis K-2021">{{Cite journal |last=Eaglesfield R, Tokatlidis K |date=2021 |title=Targeting and Insertion of Membrane Proteins in Mitochondria |journal=Frontiers in Cell and Developmental Biology |volume=9 |page=803205 |doi=10.3389/fcell.2021.803205 |pmid=35004695 |pmc=8740019 |via=Frontiers|doi-access=free }}</ref><ref name="Wiedemann N, Pfanner N-2017">{{Cite journal |last=Wiedemann N, Pfanner N |date=2017 |title=Mitochondrial Machineries for Protein Import and Assembly |journal=Annual Review of Biochemistry |volume=86 |pages=685–714 |doi=10.1146/annurev-biochem-060815-014352 |pmid=28301740 |via=Ann Rev Biochem Online|doi-access=free }}</ref> Unfolded proteins bound by [[cytosolic]] [[Chaperone (protein)|chaperone]] [[hsp70]] that are targeted to the mitochondria may be localized to four different areas depending on their sequences.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Wiedemann N, Pfanner N-2017" /><ref name="Truscott K, Pfanner N, Voos W-2001">{{Cite journal |last=Truscott K, Pfanner N, Voos W |date=2001 |title=Transport of proteins into mitochondria |journal=Reviews of Physiology, Biochemistry and Pharmacology |volume=143 |pages=81–136 |doi=10.1007/BFb0115593 |pmid=11428265 |isbn=978-3-540-41474-2 }}</ref> They may be targeted to the [[mitochondrial matrix]], the outer membrane, the [[intermembrane space]], or the inner membrane. Defects in any one or more of these processes has been linked to health and disease.<ref>{{Cite journal |last=Kang Y, Fielden L, Stojanovski D |date=2018 |title=Mitochondrial protein transport in health and disease |journal=Seminars in Cell & Developmental Biology |volume=76 |pages=142–153 |doi=10.1016/j.semcdb.2017.07.028 |pmid=28765093 }}</ref>

==== Mitochondrial matrix ====
Proteins destined for the mitochondrial matrix have specific signal sequences at their beginning (N-terminus) that consist of a string of 20 to 50 amino acids. These sequences are designed to interact with receptors that guide the proteins to their correct location inside the mitochondria. The sequences have a unique structure with clusters of water-loving (hydrophilic) and water-avoiding (hydrophobic) amino acids, giving them a dual nature known as amphipathic. These amphipathic sequences typically form a spiral shape (alpha-helix) with the charged amino acids on one side and the hydrophobic ones on the opposite side. This structural feature is essential for the sequence to function correctly in directing proteins to the matrix. If mutations occur that mess with this dual nature, the protein often fails to reach its intended destination, although not all changes to the sequence have this effect. This indicates the importance of the amphipathic property for the protein to be correctly targeted to the mitochondrial matrix.<ref name="Lodish-2016" />[[File:Inner membrane and matrix protein targeting.png|thumb|The pre-sequence pathway into the mitochondrial inner membrane (IM) and mitochondrial matrix.]]Proteins targeted to the mitochondrial matrix first involves interactions between the matrix targeting sequence located at the N-terminus and the outer membrane import receptor complex TOM20/22.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Araiso Y, Endo T-2022" /><ref name="Pfanner N, Geissler A-2001">{{Cite journal |last=Pfanner N, Geissler A |date=2001 |title=Versatility of the mitochondrial protein import machinery |url=https://www.nature.com/articles/35073006 |journal=Nature Reviews Molecular Cell Biology |volume=2 |issue=5 |pages=339–349 |doi=10.1038/35073006 |pmid=11331908 |s2cid=21011113 |via=Nature|url-access=subscription }}</ref> In addition to the docking of internal sequences and [[cytosolic]] [[Chaperone (protein)|chaperones]] to TOM70.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Araiso Y, Endo T-2022" /><ref name="Pfanner N, Geissler A-2001" /> Where TOM is an abbreviation for translocase of the outer membrane. Binding of the matrix targeting sequence to the import receptor triggers a handoff of the polypeptide to the general import core (GIP) known as TOM40.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Araiso Y, Endo T-2022" /><ref name="Pfanner N, Geissler A-2001" /> The general import core (TOM40) then feeds the polypeptide chain through the intermembrane space and into another translocase complex TIM17/23/44 located on the inner mitochondrial membrane.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Eaglesfield R, Tokatlidis K-2021" /><ref name="Wiedemann N, Pfanner N-2017" /><ref name="Bauer M, Hofmann S, Neupert W, Brunner M-2000">{{Cite journal |last=Bauer M, Hofmann S, Neupert W, Brunner M |date=2000 |title=Protein translocation into mitochondria: the role of TIM complexes |journal=Trends in Cell Biology |volume=10 |issue=1 |pages=25–31 |doi=10.1016/S0962-8924(99)01684-0 |pmid=10603473 |via=Elsevier Science Direct}}</ref> This is accompanied by the necessary release of the [[cytosolic]] [[Chaperone (protein)|chaperones]] that maintain an unfolded state prior to entering the mitochondria. As the polypeptide enters the matrix, the signal sequence is cleaved by a processing [[Protease|peptidase]] and the remaining sequences are bound by mitochondrial chaperones to await proper folding and activity.<ref name="Wiedemann N, Pfanner N-2017" /><ref name="Truscott K, Pfanner N, Voos W-2001" /> The push and pull of the polypeptide from the cytosol to the intermembrane space and then the matrix is achieved by an [[electrochemical gradient]] that is established by the mitochondrion during [[oxidative phosphorylation]].<ref name="Nelson-2017" /><ref name="Eaglesfield R, Tokatlidis K-2021" /><ref name="Wiedemann N, Pfanner N-2017" /><ref name="Truscott K, Pfanner N, Voos W-2001" /> In which a mitochondrion active in [[metabolism]] has generated a [[Membrane potential|negative potential]] inside the matrix and a [[Membrane potential|positive potential]] in the intermembrane space.<ref name="Wiedemann N, Pfanner N-2017" /><ref>{{Cite book |last=Nelson D, Cox M |title=Principles of Biochemistry |publisher=W.H. Freeman and Company |year=2017 |isbn=978-1-4641-2611-6 |edition=7th |location=New York, NY}}</ref> It is this negative potential inside the matrix that directs the positively charged regions of the targeting sequence into its desired location.

==== Mitochondrial inner membrane ====
Targeting of mitochondrial proteins to the inner membrane may follow 3 different pathways depending upon their overall sequences, however, entry from the outer membrane remains the same using the import receptor complex TOM20/22 and TOM40 general import core.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Eaglesfield R, Tokatlidis K-2021" /> The first pathway for proteins targeted to the inner membrane follows the same steps as those designated to the matrix where it contains a matrix targeting sequence that channels the polypeptide to the inner membrane complex containing the previously mentioned translocase complex TIM17/23/44.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Eaglesfield R, Tokatlidis K-2021" /><ref name="Wiedemann N, Pfanner N-2017" /> However, the difference is that the peptides that are designated to the inner membrane and not the matrix contain an upstream sequence called the stop-transfer-anchor sequence.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /> This stop-transfer-anchor sequence is a hydrophobic region that embeds itself into the [[Lipid bilayer|phospholipid bilayer]] of the inner membrane and prevents translocation further into the mitochondrion.<ref name="Eaglesfield R, Tokatlidis K-2021" /><ref name="Wiedemann N, Pfanner N-2017" /> The second pathway for proteins targeted to the inner membrane follows the matrix localization pathway in its entirety. However, instead of a stop-transfer-anchor sequence, it contains another sequence that interacts with an inner membrane protein called Oxa-1 once inside the matrix that will embed it into the inner membrane.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Eaglesfield R, Tokatlidis K-2021" /><ref name="Wiedemann N, Pfanner N-2017" /> The third pathway for mitochondrial proteins targeted to the inner membrane follow the same entry as the others into the outer membrane, however, this pathway utilizes the translocase complex TIM22/54 assisted by complex TIM9/10 in the intermembrane space to anchor the incoming peptide into the membrane.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Eaglesfield R, Tokatlidis K-2021" /><ref name="Wiedemann N, Pfanner N-2017" /> The peptides for this last pathway do not contain a matrix targeting sequence, but instead contain several internal targeting sequences.

==== Mitochondrial intermembrane space ====
If instead the precursor protein is designated to the intermembrane space of the mitochondrion, there are two pathways this may occur depending on the sequences being recognized. The first pathway to the intermembrane space follows the same steps for an inner membrane targeted protein. However, once bound to the inner membrane the [[C-terminus]] of the anchored protein is cleaved via a peptidase that liberates the preprotein into the intermembrane space so it can fold into its active state.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Wiedemann N, Pfanner N-2017" /> One of the greatest examples for a protein that follows this pathway is [[Cytochrome b|cytochrome b2]], that upon being cleaved will interact with a [[heme]] cofactor and become active.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref>{{Cite journal |last=Shwarz E, Seytter T, Guiard B, Neupert W |date=1993 |title=Targeting of cytochrome b2 into the mitochondrial intermembrane space: specific recognition of the sorting signal. |journal=EMBO J |volume=12 |via=PubMed}}</ref> The second intermembrane space pathway does not utilize any inner membrane complexes and therefor does not contain a matrix targeting signal. Instead, it enters through the general import core TOM40 and is further modified in the intermembrane space to achieve its active conformation. TIM9/10 is an example of a protein that follows this pathway in order to be in the location it needs to be to assist in inner membrane targeting.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Wiedemann N, Pfanner N-2017" /><ref>{{Cite journal |last=Kurz M, Martin H, Rassow J, Pfanner N, Ryan M |date=2017 |title=Biogenesis of Tim Proteins of the Mitochondrial Carrier Import Pathway: Differential Targeting Mechanisms and Crossing Over with the Main Import Pathway |journal=Molecular Biology of the Cell |volume=10 |issue=7 |pages=2461–2474 |doi=10.1091/mbc.10.7.2461 |pmid=10397776 |pmc=25469 }}</ref>

==== Mitochondrial outer membrane ====
Outer membrane targeting simply involves the interaction of precursor proteins with the outer membrane translocase complexes that embeds it into the membrane via internal-targeting sequences that are to form hydrophobic [[Alpha helix|alpha helices]] or [[beta barrel]]s that span the phospholipid bilayer.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /><ref name="Eaglesfield R, Tokatlidis K-2021" /><ref name="Wiedemann N, Pfanner N-2017" /> This may occur by two different routes depending on the preprotein internal sequences. If the preprotein contains internal hydrophobic regions capable of forming alpha helices, then the preprotein will utilize the mitochondrial import complex (MIM) and be transferred laterally to the membrane.<ref name="Eaglesfield R, Tokatlidis K-2021" /><ref name="Wiedemann N, Pfanner N-2017" /> For preproteins containing hydrophobic internal sequences that correlate to beta-barrel forming proteins, they will be imported from the aforementioned outer membrane complex TOM20/22 to the intermembrane space. In which they will interact with TIM9/10 intermembrane-space protein complex that transfers them to [[sorting and assembly machinery]] (SAM) that is present in the outer membrane that laterally displaces the targeted protein as a beta-barrel.<ref name="Eaglesfield R, Tokatlidis K-2021" /><ref name="Wiedemann N, Pfanner N-2017" />