Name: mitochondrial protein sorting pathways
Uploaded: 2023-04-30T09:41:52.000Z
Duration: 1 min 19 s
Channel: JoVE

Peptides carrying presequences targeted to the matrix are threaded through the TOM/TIM complex. Mitochondrial Hsp70 binds the peptide as it exits the TIM channel and moves it into the matrix, where the presequence is cleaved by matrix proteases.
Proteins targeted to the intermembrane space carry an additional hydrophobic signal sequence that stops translocation across the TIM complex. Signal peptidases cleave the hydrophobic segment and release the active protein in the intermembrane space.
Alternatively, mitochondrial intermembrane space assembly 40 or Mia40 protein imports intermembrane space proteins lacking the presequences.
Oxidized Mia40 forms a transient disulfide bond with the thiols on the incoming polypeptide and pulls the nascent peptide through the TOM channel. Once the entire peptide is threaded through, Mia40 is reduced.
Some inner membrane proteins with stop-transfer sequences are arrested and inserted into the membrane. Others are first processed by signal peptidases, and then recognized by an OXA&#160; translocase, which embeds them in the membrane.

translocation of proteins into the mitochondria

Translocation of Proteins into the Mitochondria

Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast, alpha-helical membrane-anchored proteins are translocated by the TOM complex and inserted by the mitochondrial import complex.
Sorting of intermembrane space proteins:
Intermembrane space proteins are sorted via the mitochondrial intermembrane space import and assembly machinery or (MIA) pathway. MIA40 is an oxidoreductase of the intermembrane space (IMS) that recognizes and binds specific cysteine residues upstream or downstream of the hydrophobic intermembrane space sorting sequence called the MISS/ITS signal. MIA40 contains a redox-active CPC motif that facilitates thiol-disulfide exchange with the cysteine residues of the incoming precursor. Dimeric flavin-dependent oxidoreductase called Erv1 cooperates with MIA40 in oxidizing the precursor cysteine residues, thereby folding and transporting the oxidized precursor into the IMS. Erv1 then transfers electrons to the reduced CPC motif of MIA40 via cytochrome C and cytochrome C oxidase or respiratory chain IV of the electron transport chain. Oxidized Mia40 gets ready for another round of IMS precursor import.
Sorting of inner membrane and matrix proteins:
Inner membrane proteins with cleavable N terminal presequences are translocated through the presequence translocase TIM23 assisted by the ATP-dependent presequence translocase associated motor (PAM). In contrast, inner membrane proteins with internal import signals called carrier signatures are translocated via the carrier translocase TIM22.&#160; Translocation across TIM23 or TIM22 to the inner membrane can follow two distinct routes of sorting: the conservative and stop transport pathways. In the stop-transfer pathway, the movement of precursors with internal hydrophobic patches gets blocked across the TIM22/23 channel, which gets laterally released onto the inner membrane. In the conservative pathway, precursors are first translocated to the matrix and then assembled into the inner membrane by the Oxa protein. However, precursors targeted to the matrix are cleaved by matrix processing peptidases. Some precursors are further processed by mitochondrial intermediate peptidase or inner membrane protease I depending on additional presequence cleavage sites. Processed matrix proteins are then released into the matrix to be folded by chaperonins using ATP hydrolysis energy.

Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called ...

translocation-of-proteins-into-the-mitochondria

Education

JoVE Core

Cell Biology

Unit 1

Intracellular Compartments and Protein Sorting

intracellular-compartments-and-protein-sorting

KEY TERMS AND CONCEPTS

mitochondrial protein sorting pathways

Mitochondrial protein sorting pathways move precursor proteins to the outer membrane, intermembrane space, inner membrane, or matrix. These proteins use different translocases, which are protein machines that guide them to the right mitochondrial compartment.

Outer membrane proteins follow two routes. Beta-barrel porins pass through the TOM complex and are inserted into the outer membrane by the SAM complex. Alpha-helical, membrane-anchored proteins also pass through TOM, but they are inserted by the mitochondrial import complex.

Proteins in the intermembrane space use the MIA pathway, which stands for mitochondrial intermembrane space import and assembly. MIA40 is an oxidoreductase in the intermembrane space. It recognizes cysteine residues near a hydrophobic MISS/ITS sorting signal and uses its redox-active CPC motif to help form disulfide bonds with incoming precursor proteins. Erv1, a dimeric flavin-dependent oxidoreductase, works with MIA40 to oxidize these precursors, fold them, and move them into the intermembrane space. Erv1 then passes electrons through cytochrome C and cytochrome C oxidase, also called respiratory chain IV of the electron transport chain, to reduce the CPC motif so MIA40 can start another round.

Inner membrane and matrix proteins use TIM23 or TIM22. Proteins with cleavable N-terminal presequences move through TIM23 with help from the ATP-dependent PAM motor. Proteins with internal carrier signatures use TIM22 instead. Two sorting routes are used after entry. In the stop-transfer pathway, a hydrophobic region blocks further movement, and the protein is released into the inner membrane. In the conservative pathway, the precursor first reaches the matrix and is later assembled into the inner membrane by Oxa.

Matrix-targeted precursors are cleaved by matrix processing peptidases. Some proteins are processed further by mitochondrial intermediate peptidase or inner membrane protease I if extra cleavage sites are present. After processing, matrix proteins are released into the matrix and folded by chaperonins. This folding step uses energy from ATP hydrolysis.

Mitochondrial Protein Sorting Pathways

overview of protein sorting and transport

overview-of-protein-sorting-and-transport

Protein Targeting Inside Eukaryotic Cells

Protein targeting helps eukaryotic cells send proteins to the right organelle. Each membrane-bound organelle has its own protein needs, so proteins must be sorted after they are made. This process is called protein sorting. Protein sorting happens in two main ways: signal-based sorting and vesicle-based trafficking. Signal-based sorting uses sorting signals, which are specific amino acid sequences. These signals direct proteins to the correct place in the cell through gated transport or...

signal sequences and sorting receptors

Protein Targeting Signals and Sorting Receptors

Protein targeting signals help newly made proteins reach the correct place inside a cell. These short amino acid sequences act like address labels. They guide proteins to the organelle where they belong. Classical signal sequences are usually 15 to 60 amino acids long. They are found at the N-terminus, or beginning, of a polypeptide chain. These signals often include a basic region near the start, a hydrophobic core, and a polar C-terminus. The C-terminus also contains a signal cleavage site...

nuclear protein sorting

How Proteins Cross the Nuclear Envelope

Nuclear protein sorting controls how proteins, RNA, and ribosomes move between the nucleus and the cytosol. Histones, polymerases, and gene regulatory proteins are carried into the nucleus. RNA and ribosomes are exported to the cytosol. This selective transport helps regulate gene expression inside the cell. Proteins that enter the nucleus usually carry a nuclear localization signal, or NLS. Import receptors in the cytosol recognize this signal and bind the cargo. Proteins that leave the...

nuclear localization signals and import

Nuclear Import Signals in Proteins

Proteins that need to enter the nucleus carry a nuclear localization signal, or NLS. This short amino acid stretch helps the cell sort proteins to the right place. In many proteins, the signal is made of positively charged amino acids such as lysine and arginine and can appear as a loop or a signal patch. Classical nuclear localization signals have two main forms. A monopartite cNLS is a single cluster of 4 to 8 amino acids. A bipartite cNLS has two clusters of 2 to 3 amino acids with a...

nuclear export

Nuclear Export Signals and Cargo

Nuclear export signals help proteins and RNA leave the nucleus and reach the right place in the cell. The nucleus also keeps some proteins inside by using a nuclear retention sequence, or NRS, which anchors them to the nuclear lamins and blocks movement to the cytosol. Proteins that are not restricted can move after synthesis to the cytosol or to other organelles. They do this with the help of nuclear export signals, or NES. The transcript describes three NES types: the canonical leucine-rich...

directionality of nuclear transport

Ran GTP Gradient in Nuclear Transport

Ran GTP helps control the direction of nuclear transport in cells. Ran, or Ras-related nuclear protein, is a small G protein that switches between GTP-bound and GDP-bound forms. Its activity depends on two key regulators: RanGAP in the cytosol and RanGEF inside the nucleus. A higher RanGTP level builds up inside the nucleus because GTP is abundant in cells and Ran regulators are unevenly distributed. This RanGTP gradient across the nuclear envelope supports one-way movement of nuclear proteins.

regulation of nuclear protein sorting

Nuclear Transport Controls Protein Entry

Nuclear transport controls which proteins enter and leave the nucleus, and it helps shape gene expression in eukaryotic cells. Because the nucleus must keep a specific composition, movement across the nuclear envelope is tightly regulated. Cells can slow or stop this transport in several ways. One common control is to hide the protein’s sorting signal. Nuclear receptors first bind nuclear localization signals, or NLS, and nuclear export signals, or NES. A cargo protein can change shape through...

mitochondrial protein sorting

Mitochondrial Protein Import Routes

Mitochondrial protein import routes move proteins to the right place inside mitochondria. Mitochondria are double-membrane organelles in eukaryotes. They support cellular metabolism, signaling, ATP synthesis, and programmed cell death. Each of these jobs depends on proteins and enzymes reaching the correct mitochondrial subcompartment. Most mitochondrial proteins are made in the nucleus and then imported into the organelle as unfolded or loosely folded precursors. These precursors usually...

mitochondrial precursor proteins

How Mitochondria Recognize Protein Precursors

Mitochondria recognize precursor proteins before they enter the organelle. These precursors are newly made polypeptide chains that stay partially unfolded or loosely folded. Cytosolic chaperones, including heat shock protein 70 (Hsp70), and mitochondrial import stimulation factors (MSFs) help keep them from folding too soon. Precursors bound to MSFs are directed to the TOM70-TOM37 receptor complex. Precursors bound to Hsp70 chaperones are targeted to the TOM20-TOM22 receptor complex. Most...

Mitochondrial protein sorting pathways move precursor proteins to the outer membrane, intermembrane space, inner membrane, or matrix. These proteins use different translocases, which are protein machines that guide them to the right mitochondrial compartment. Outer membrane proteins follow two routes. Beta-barrel porins pass through the TOM complex and are inserted into the outer membrane by the SAM complex. Alpha-helical, membrane-anchored proteins also pass through TOM, but they are inserted...

energy to drive translocation

Mitochondrial Protein Import Energy

Mitochondrial protein import uses ATP hydrolysis and an electrochemical potential across the inner membrane. Newly made precursor proteins first bind to Hsp70 chaperones in the cytosol. These chaperones help guide the proteins to import receptors on the mitochondrial surface. ATP hydrolysis gives Hsp70 the energy to pass precursors to the TOM receptors on the outer membrane. TOM and TIM are the main import complexes that move proteins across the two mitochondrial membranes. Before transport,...

structure of porins

Porin Assembly in Mitochondria

Porin assembly in mitochondria depends on beta-barrel proteins that move across the outer membrane and fold into their final form. Mitochondria, chloroplasts, and gram-negative bacteria use these transmembrane proteins to let ions and metabolites diffuse freely across membranes. Mitochondrial porin precursors carry conserved beta signals at their C-terminal end. These beta signals contain the PoXGXXHyXHy motif, where Po is polar, X is any amino acid, G is glycine, and Hy is a large hydrophobic...

porin insertion in the outer mitochondrial membrane

porin-insertion-in-the-outer-mitochondrial-membrane

SAM Complex Assembly of Mitochondrial Porins

Mitochondrial porins are beta-barrel proteins that are built into the outer membrane with help from the TOM and SAM systems. Porin precursors first move through the TOM complex into the intermembrane space. They then bind TIM chaperones, which guide them to the Sorting and Assembly Machinery, or SAM, complex on the outer mitochondrial membrane. The SAM complex helps fold and place porins into the membrane, but the exact assembly route is still modeled in different ways. In one model, the porin...

protein transport into the inner mitochondrial membrane

protein-transport-into-the-inner-mitochondrial-membrane

Mitochondrial Import Pathways to the Inner Membrane

Mitochondrial import pathways move nuclear-encoded precursor proteins to the inner membrane in several steps. Two main translocons, TIM22 and TIM23, handle much of this sorting. A third translocase, OXA, helps insert some proteins into the membrane after they have moved partway through the system. TIM23 is a cation-selective pore, which means it favors positively charged signals. It stays closed by the N-terminal segment of the protein. Negative charges on TIM23 act like a receptor for the...

protein transport to the thylakoids

Thylakoid Protein Targeting Pathways

Thylakoid protein targeting pathways move precursor proteins to the chloroplast thylakoids, where photosynthesis takes place. Thylakoids are membrane-bound sac-like structures inside the chloroplast. Their lumen holds many electron transport proteins, and their membrane is rich in the light-harvesting complex. Proteins destined for the thylakoids first enter through the general TOC/TIC import pathway. In the stroma, stromal processing peptidases remove the transit signal and expose thylakoid...

protein transport to the stroma

Chloroplast Protein Import into the Stroma

Chloroplast protein import into the stroma depends on a set of membrane systems and protein machines that place each protein in the right location. Chloroplasts have an outer membrane, an inner membrane, and a thylakoid membrane. Each one contains its own transporters, translocons, and enzymes, so sorting is essential for chloroplast function. Two major complexes carry out this import step. The translocon of the outer chloroplast membrane, or TOC complex, works with the translocon of the inner...

protein transport to the outer chloroplast membrane

protein-transport-to-the-outer-chloroplast-membrane

Chloroplast Outer Membrane Protein Import

Chloroplast outer membrane protein import begins in the cytosol, where nucleus-encoded proteins are made. After synthesis, these precursors bind cytosolic factors such as 14-3-3 protein and Hsp70 chaperones. These factors keep the proteins unfolded until they can move to the chloroplast. Two models explain how the precursor is recognized by the TOC complex, which is the outer membrane transport system. In the first model, the new precursor binds TOC159 and forms a complex. That complex docks...

protein transport to the inner chloroplast membrane

protein-transport-to-the-inner-chloroplast-membrane

Inner Chloroplast Membrane Protein Pathways

Proteins reach the inner chloroplast membrane through several import pathways. These proteins, often called plastid proteins, use N-terminal transit sequences and internal targeting sequences to reach the correct chloroplast subcompartment. One route is the stop-transfer pathway. In this pathway, an internal hydrophobic sequence blocks movement into the stroma, the fluid-filled space inside the chloroplast. The precursor pauses across the TIC complex and is then released sideways into the...

key terms and concepts

Learn how mitochondrial proteins are sorted into the outer membrane, intermembrane space, inner membrane, and matrix using TOM, TIM, SAM, MIA, and Oxa.

Mitochondrial Protein Sorting Pathways | JoVE K12

cell biology

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Mitochondrial Protein Sorting Pathways

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