Editing Protein targeting (section)

==Sorting of proteins==
=== 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" />

===Chloroplasts===
[[Chloroplast]]s are similar to mitochondria in that they contain their own DNA for production of some of their components. However, the majority of their proteins are obtained via post-translational translocation and arise from nuclear genes. Proteins may be targeted to several sites of the chloroplast depending on their sequences such as the outer envelope, inner envelope, stroma, thylakoid lumen, or the thylakoid membrane.<ref name="Lodish, Berk, Kaiser, Krieger, Bretscher, Ploegh, Martin, Yaffe, Amon-2021" /> Proteins are targeted to Thylakoids by mechanisms related to Bacterial Protein Translocation.<ref name="Lodish-2016">{{Cite book |last1=Lodish |first1=Harvey |title=Molecular Cell Biology |last2=Berk |first2=Arnold |last3=Kaiser |first3=Chris A |last4=Krieger |first4=Monty |last5=Bretscher |first5=Anthony |last6=Ploegh |first6=Hidde |last7=Amon |first7=Angelika |last8=Scott |first8=Matthew P |publisher=W.H. Freeman and Company |year=2016 |isbn=978-1-4641-8339-3 |edition=8th |location=New York, USA |pages=609–610 |language=en}}</ref> Proteins targeted to the envelope of chloroplasts usually lack cleavable sorting sequence and are laterally displaced via membrane sorting complexes. General import for the majority of preproteins requires translocation from the cytosol through the [[TIC/TOC complex|Toc and Tic complexes]] located within the chloroplast envelope. Where Toc is an abbreviation for the translocase of the outer chloroplast envelope and Tic is the translocase of the inner chloroplast envelope. There is a minimum of three proteins that make up the function of the Toc complex. Two of which, referred to as Toc159 and Toc34, are responsible for the docking of stromal import sequences and both contain [[GTPase]] activity. The third known as Toc 75, is the actual translocation channel that feeds the recognized preprotein by Toc159/34 into the chloroplast.<ref name="Soll J, Schleiff E-2004">{{Cite journal |last=Soll J, Schleiff E |date=2004 |title=Protein import into chloroplasts |url=https://www.nature.com/articles/nrm1333 |journal=Nature Reviews Molecular Cell Biology |volume=5 |issue=3 |pages=198–208 |doi=10.1038/nrm1333 |pmid=14991000 |s2cid=32453554 |via=Nature}}</ref>

==== Stroma ====
Targeting to the stroma requires the preprotein to have a stromal import sequence that is recognized by the Tic complex of the inner envelope upon being translocated from the outer envelope by the Toc complex. The Tic complex is composed of at least five different Tic proteins that are required to form the translocation channel across the inner envelope.<ref>{{Cite journal |last=Gutensohn M, Fan E, Frielingsdorf S, Hanner P, Hou B, Hust B, Klosgen R |date=2005 |title=Toc, Tic, Tat et al.: structure and function of protein transport machineries in chloroplasts |url=https://pubmed.ncbi.nlm.nih.gov/16386331/ |journal=Journal of Plant Physiology |volume=163 |issue=3 |pages=333–347 |doi=10.1016/j.jplph.2005.11.009 |pmid=16386331 |via=PubMed}}</ref> Upon being delivered to the stroma, the stromal import sequence is cleaved off via a signal peptidase. This delivery process to the stroma is currently known to be driven by [[ATP hydrolysis]] via stromal [[Heat shock protein|HSP]] chaperones, instead of the transmembrane [[electrochemical gradient]] that is established in mitochondria to drive protein import.<ref name="Soll J, Schleiff E-2004" /> Further intra-chloroplast sorting depends on additional target sequences such as those designated to the [[Thylakoid|thylakoid membrane]] or the [[Thylakoid|thylakoid lumen]].

==== Thylakoid lumen ====
If a protein is to be targeted to the thylakoid lumen, this may occur via four differently known routes that closely resemble bacterial protein transport mechanisms. The route that is taken depends upon the protein delivered to the stroma being in either an unfolded or metal-bound folded state. Both of which will still contain a thylakoid targeting sequence that is also cleaved upon entry to the lumen. While protein import into the stroma is ATP-driven, the pathway for metal-bound proteins in a folded state to the thylakoid lumen has been shown to be driven by a pH gradient.[[File:Protein targeting to the thylakoid membrane in chloroplasts.png|thumb|Pathways for proteins targeted to the thylakoid membrane in chloroplasts.]]

==== Thylakoid membrane ====
Proteins bound for the membrane of the thylakoid will follow up to four known routes that are illustrated in the corresponding figure shown. They may follow a co-translational insertion route that utilizes stromal ribosomes and the SecY/E transmembrane complex, the SRP-dependent pathway, the spontaneous insertion pathway, or the GET pathway. The last of the three are post-translational pathways originating from nuclear genes and therefor constitute the majority of proteins targeted to the thylakoid membrane. According to recent review articles in the journal of biochemistry and molecular biology, the exact mechanisms are not yet fully understood.

=== Both chloroplasts and mitochondria ===
Many proteins are needed in both mitochondria and chloroplasts.<ref>{{cite journal | vauthors = Sharma M, Bennewitz B, Klösgen RB | title = Rather rule than exception? How to evaluate the relevance of dual protein targeting to mitochondria and chloroplasts | journal = Photosynthesis Research | volume = 138 | issue = 3 | pages = 335–343 | date = December 2018 | pmid = 29946965 | doi = 10.1007/s11120-018-0543-7 | bibcode = 2018PhoRe.138..335S | s2cid = 49427254 }}</ref> In general the dual-targeting peptide is of intermediate character to the two specific ones. The targeting peptides of these [[protein]]s have a high content of basic and [[hydrophobic]] [[amino acids]], a low content of negatively charged [[amino acids]]. They have a lower content of alanine and a higher content of leucine and phenylalanine. The dual targeted proteins have a more hydrophobic targeting peptide than both mitochondrial and chloroplastic ones. However, it is tedious to predict if a peptide is dual-targeted or not based on its [[Physiochemical|physio-chemical]] characteristics.

=== Nucleus ===
The nucleus of a cell is surrounded by a nuclear envelope consisting of two layers, with the inner layer providing structural support and anchorage for chromosomes and the nuclear lamina.<ref name="Alberts-2018" /> The outer layer is similar to the endoplasmic reticulum (ER) membrane. This envelope contains nuclear pores, which are complex structures made from around 30 different proteins.<ref name="Alberts-2018" /> These pores act as selective gates that control the flow of molecules into and out of the nucleus.

While small molecules can pass through these pores without issue, larger molecules, like RNA and proteins destined for the nucleus, must have specific signals to be allowed through.<ref name="Lodish-2008" /> These signals are known as nuclear localization signals, usually comprising short sequences rich in positively charged amino acids like lysine or arginine.<ref name="Alberts-2018" /> 

Proteins called nuclear import receptors recognize these signals and guide the large molecules through the nuclear pores by interacting with the disordered, mesh-like proteins that fill the pore.<ref name="Alberts-2018" /> The process is dynamic, with the receptor moving the molecule through the meshwork until it reaches the nucleus.<ref name="Lodish-2008" />

Once inside, a GTPase enzyme called Ran, which can exist in two different forms (one bound to GTP and the other to GDP), facilitates the release of the cargo inside the nucleus and recycles the receptor back to the cytosol.<ref name="Alberts-2018" /><ref name="Lodish-2008" /> The energy for this transport comes from the hydrolysis of GTP by Ran. Similarly, nuclear export receptors help move proteins and RNA out of the nucleus using a different signal and also harnessing Ran's energy conversion.<ref name="Alberts-2018" />

Overall, the nuclear pore complex works efficiently to transport macromolecules at high speed, allowing proteins to move in their folded state and ribosomal components as complete particles, which is distinct from how proteins are transported into most other organelles.<ref name="Alberts-2018" />

=== Endoplasmic reticulum ===
The endoplasmic reticulum (ER) plays a key role in protein synthesis and distribution in eukaryotic cells. It's a vast network of membranes where proteins are processed and sorted to various destinations, including the ER itself, the cell surface, and other organelles like the Golgi apparatus, endosomes, and lysosomes.<ref name="Alberts-2018" /> Unlike other organelle-targeted proteins, those headed for the ER start to be transferred across its membrane while they're still being made.<ref name="Lodish-2008" /><ref name="Alberts-2018" />

==== Protein synthesis and sorting  ====
There are two types of proteins that move to the ER: water-soluble proteins, which completely cross into the ER lumen, and transmembrane proteins, which partly cross and embed themselves within the ER membrane.<ref name="Lodish-2008" /> These proteins find their way to the ER with the help of an ER signal sequence, a short stretch of hydrophobic amino acids.<ref name="Alberts-2018" />

Proteins entering the ER are synthesized by ribosomes. There are two sets of ribosomes in the cell: those bound to the ER (making it look 'rough') and those floating freely in the cytosol. Both sets are identical but differ in the proteins they synthesize at a given moment.<ref name="Alberts-2018" /><ref name="Lodish-2008" /> Ribosomes that are making proteins with an ER signal sequence attach to the ER membrane and start the translocation process. This process is energy-efficient because the growing protein chain itself pushes through the ER membrane as it elongates.<ref name="Alberts-2018" />

As the mRNA is translated into a protein, multiple ribosomes may attach to it, creating a structure called a polyribosome.<ref name="Alberts-2018" /> If the mRNA is coding for a protein with an ER signal sequence, the polyribosome attaches to the ER membrane, and the protein begins to enter the ER while it is still being synthesized.<ref name="Lodish-2008" /><ref name="Alberts-2018" />

===== Guided entry of soluble proteins =====
In the process of protein synthesis within eukaryotic cells, soluble proteins that are destined for the endoplasmic reticulum (ER) or for secretion out of the cell are guided to the ER by a two-part system. Firstly, a signal-recognition particle (SRP) in the cytosol attaches to the emerging protein's ER signal sequence and the ribosome itself.<ref name="Alberts-2018" /> Secondly, an SRP receptor located in the ER membrane recognizes and binds to the SRP. This interaction temporarily slows down protein synthesis until the SRP and ribs complex binds to the SRP receptor on the ER.<ref name="Alberts-2018" /><ref name="Lodish-2008" />

Once this binding occurs, the SRP is released, and the ribosome is transferred to a protein translocator in the ER membrane, allowing protein synthesis to continue.<ref name="Alberts-2018" /><ref name="Lodish-2008" /> The polypeptide chain of the protein is then threaded through a channel in the translocator into the ER lumen. The signal sequence of the protein, typically at the beginning (N-terminus) of the polypeptide chain, plays a dual role. It not only targets the ribosome to the ER but also triggers the opening of the translocator.<ref name="Alberts-2018" /> As the protein is fed through the translocator, the signal sequence stays attached, allowing the rest of the protein to move through as a loop. A signal peptidase inside the ER then cuts off the signal sequence, which is subsequently discarded into the lipid bilayer of the ER membrane and broken down.<ref name="Alberts-2018" /><ref name="Lodish-2008" />

Finally, once the last part of the protein (the C-terminus) passes through the translocator, the entire soluble protein is released into the ER lumen, where it can then fold and undergo further modifications or be transported to its final destination.<ref name="Alberts-2018" /><ref name="Lodish-2008" />

====== Mechanisms of transmembrane protein integration ======
Transmembrane proteins, which are partly integrated into the ER membrane rather than released into the ER lumen, have a complex assembly process.<ref name="Alberts-2018" /><ref name="Lodish-2008" /> The initial stages are similar to soluble proteins: a signal sequence starts the insertion into the ER membrane. However, this process is interrupted by a stop-transfer sequence—a string of hydrophobic amino acids—which causes the translocator to halt and release the protein laterally into the membrane.<ref name="Alberts-2018" /><ref name="Lodish-2008" /> This results in a single-pass transmembrane protein with one end inside the ER lumen and the other in the cytosol, and this orientation is permanent.<ref name="Alberts-2018" />

Some transmembrane proteins use an internal signal (start-transfer sequence) instead of one at the N-terminus, and unlike the initial signal sequence, this start-transfer sequence isn't removed.<ref name="Alberts-2018" /><ref name="Lodish-2008" /> It begins the transfer process, which continues until a stop-transfer sequence is encountered, at which point both sequences become anchored in the membrane as alpha-helical segments.<ref name="Alberts-2018" />

In more complex proteins that span the membrane multiple times, additional pairs of start- and stop-transfer sequences are used to weave the protein into the membrane in a fashion akin to a sewing machine. Each pair allows a new segment to cross the membrane and adds to the protein's structure, ensuring it is properly embedded with the correct arrangement of segments inside and outside the ER membrane.<ref name="Alberts-2018" />

===Peroxisomes===
[[File:Peroxisome Protein Targeting.png|thumb|Generalized Protein Targeting to the Peroxisomal Matrix]]
[[Peroxisome]]s contain a single phospholipid bilayer that surrounds the peroxisomal matrix containing a wide variety of proteins and enzymes that participate in anabolism and catabolism. Peroxisomes are specialized cell organelles that carry out specific oxidative reactions using molecular oxygen. Their primary function is to remove hydrogen atoms from organic molecules, a process that results in the production of [[hydrogen peroxide]] ({{chem2|H2O2}}).<ref name="Alberts B, Johnson A, Lewis J-2002" /><ref name="Lodish-2008" /> Within peroxisomes, an enzyme called [[catalase]] plays a critical role. It uses the hydrogen peroxide generated in the earlier reaction to oxidize various other substances, including [[phenols]], [[formic acid]], [[formaldehyde]], and alcohol.<ref name="Alberts B, Johnson A, Lewis J-2002" /><ref name="Lodish-2008" /> This is known as the "peroxidative" reaction.<ref name="Lodish-2008" />

Peroxisomes are particularly important in liver and kidney cells for detoxifying harmful substances that enter the bloodstream. For example, they are responsible for oxidizing about 25% of the [[ethanol]] we consume into [[acetaldehyde]].<ref name="Alberts B, Johnson A, Lewis J-2002" /> Additionally, catalase within peroxisomes can break down excess hydrogen peroxide into water and oxygen and thus preventing potential damage from the build-up of {{chem2|H2O2}}.<ref name="Alberts B, Johnson A, Lewis J-2002" /><ref name="Lodish-2008" /> Since it contains no internal DNA like that of the mitochondria or chloroplast all [[peroxisome|peroxisomal]] proteins are encoded by nuclear genes.<ref>{{Cite book|url=https://www.worldcat.org/oclc/828743403|title=Encyclopedia of biological chemistry|others=Lennarz, William J.,, Lane, M. Daniel|date=8 January 2013|isbn=978-0-12-378631-9|edition=Second|location=London|oclc=828743403}}</ref> To date there are two types of known [[Peroxisomal targeting signal|Peroxisome Targeting Signals]] (PTS):

# '''Peroxisome targeting signal 1 (PTS1)''': a C-terminal tripeptide with a consensus sequence (S/A/C)-(K/R/H)-(L/A). The most common PTS1 is [[serine]]-[[lysine]]-[[leucine]] (SKL).<ref name="Alberts B, Johnson A, Lewis J-2002">{{Cite book |last=Alberts B, Johnson A, Lewis J |title=Molecular Biology of the Cell |publisher=Garland Science |year=2002 |edition=4th |location=New York, NY}}</ref> The initial research that led to the discovery of this consensus observed that when firefly luciferase was expressed in cultured insect cells it was targeted to the peroxisome. By testing a variety of mutations in the gene encoding the expressed [[luciferase]], the consensus sequence was then determined.<ref>{{Cite journal |last=Keller G, Gould S, Deluca M, Subramani S |date=1987 |title=Firefly luciferase is targeted to peroxisomes in mammalian cells |journal=Proceedings of the National Academy of Sciences |volume=84 |issue=10 |pages=3264–3268 |doi=10.1073/pnas.84.10.3264 |pmid=3554235 |pmc=304849 |bibcode=1987PNAS...84.3264K |doi-access=free }}</ref> It has also been found that by adding this C-terminal sequence of SKL to a cytosolic protein that it becomes targeted for transport to the peroxisome. The majority of peroxisomal matrix proteins possess this PTS1 type signal.
# '''Peroxisome targeting signal 2 (PTS2)''': a nonapeptide located near the N-terminus with a consensus sequence (R/K)-(L/V/I)-XXXXX-(H/Q)-(L/A/F) (where X can be any amino acid).<ref name="Alberts B, Johnson A, Lewis J-2002" />

There are also proteins that possess neither of these signals. Their transport may be based on a so-called "piggy-back" mechanism: such proteins associate with PTS1-possessing matrix proteins and are translocated into the peroxisomal matrix together with them.<ref>{{cite journal | vauthors = Saryi NA, Hutchinson JD, Al-Hejjaj MY, Sedelnikova S, Baker P, Hettema EH | title = Pnc1 piggy-back import into peroxisomes relies on Gpd1 homodimerisation | journal = Scientific Reports | volume = 7 | issue = 1 | pages = 42579 | date = February 2017 | pmid = 28209961 | doi = 10.1038/srep42579 | pmc = 5314374 | bibcode = 2017NatSR...742579S }}</ref>

In the case of cytosolic proteins that are produced with the PTS1 C-terminal sequence, its path to the peroxisomal matrix is dependent upon binding to another cytosolic protein called [[PEX5|pex5]] (peroxin 5).<ref name="Baker A, Hogg T, Warriner S-2016">{{Cite journal |last=Baker A, Hogg T, Warriner S |date=2016 |title=Peroxisome protein import: a complex journey |journal=Biochemical Society Transactions |volume=44 |issue=3 |pages=783–789 |doi=10.1042/BST20160036 |pmid=27284042 |pmc=4900764 }}</ref> Once bound, pex5 interacts with a peroxisomal membrane protein [[PEX14|pex14]] to form a complex. When the pex5 protein with bound cargo interacts with the pex14 membrane protein, the complex induces the release of the targeted protein into the matrix. Upon releasing the cargo protein into the matrix, pex5 dissociation from pex14 occurs via [[ubiquitin]]ylation by a membrane complex comprising pex2, [[PEX12|pex12]], and [[PEX10|pex10]] followed by an ATP dependent removal involving the cytosolic protein complex [[PEX1|pex1]] and [[PEX6|pex6]].<ref>{{Cite journal |last=Gould S, Collins C |date=2002 |title=Peroxisomal-protein import: is it really that complex? |journal=Nature Rev. Mol. Cell Biol. |volume=3 |issue=5 |pages=382–389 |doi=10.1038/nrm807 |pmid=11988772 |s2cid=2522184 |doi-access=free }}</ref> The cycle for pex5 mediated import into the peroxisomal matrix is restored after the ATP dependent removal of [[ubiquitin]] and is free to bind with another protein containing a PTS1 sequence.<ref name="Baker A, Hogg T, Warriner S-2016" /> Proteins containing a PTS2 targeting sequence are mediated by a different cytosolic protein but are believed to follow a similar mechanism to that of those containing the PTS1 sequence.<ref name="Alberts B, Johnson A, Lewis J-2002" />