Name: mitochondrial protein import energy
Uploaded: 2023-04-30T09:41:59.000Z
Duration: 1 min 37 s
Channel: JoVE

Mitochondrial protein translocation is fueled by two distinct energy sources: ATP hydrolysis and the electrochemical potential across the inner membrane.
As a chaperone-bound precursor passes through the TOM complex to reach the TIM channel, an electrochemical potential of 200 millivolts across the inner membrane pulls the positively charged presequence through.
In the matrix, mitochondrial Hsp70 binds the incoming peptide and translocates it by the thermal ratchet or cross-bridge ratchet model.
In the thermal ratchet model, an emerging polypeptide chain moves back and forth across the TIM channel. When the ATP bound to Hsp70 is hydrolyzed, it prevents further polypeptide backsliding. As multiple Hsp70s bind, the precursor is translocated forward to the mitochondrial matrix.
In the cross-bridge ratchet model, matrix Hsp70 binds the TIM complex near the mouth of its channel.
As ATP binds, Hsp70 undergoes a conformational change to latch on to the precursor as it exits the TIM channel. ATP hydrolysis secures the binding of Hsp70, pulling the rest of the peptide through the TIM channel.
Rebinding of ATP triggers the dissociation of Hsp70, following which the peptide is released into t

energy to drive translocation

Energy to Drive Translocation

Mitochondrial protein import is powered by two distinct energy sources: ATP hydrolysis and electrochemical potential across the inner membrane. Newly synthesized precursors are bound by cytosolic chaperones of the Hsp70 family, which guide them to the import receptors on the mitochondrial surface. Utilizing the energy of ATP hydrolysis, Hsp70 chaperones transfer these precursors to the TOM receptors on the mitochondrial outer membrane.
Generally, polypeptides are unfolded by two distinct mechanisms before being transported in the TOM/TIM import pathway:&#160; spontaneous global unfolding and catalyzed unfolding. Precursors with shorter presequences undergo spontaneous global unfolding. In contrast, precursors with longer positively charged presequences undergo local unfolding. The unstructured presequence then traverses the TOM/TIM import complexes and interacts with the inner membrane&#8217;s negative charges to reach the matrix. Mitochondrial Hsp70 (mtHsp70) associated with TIM44 recognizes the emerging polypeptide and translocates it entirely into the matrix by accelerating the unfolding process. mtHsp70 also traps any incoming loosely folded precursors without undergoing any conformational change and translocates them to the matrix without undergoing any conformational change.
In contrast, the translocation of tightly folded polypeptides induces ATP-dependent conformational changes in mtHsp70. mtHsp70 utilizes the ATP hydrolysis energy to pull the incoming peptide across the TIM translocase. Rebinding of ATP causes the opening of mtHsp70, and the precursor gets released into the matrix.
Two models can describe precursor translocation by the mtHsp70: the thermal ratchet model and the cross-bridge ratchet model. MtHsp70 translocates precursors by trap and release mechanism in the thermal ratchet model. mtHsp70 uses the ATP hydrolysis energy to bind the spontaneously unfolded precursors and trap them into the matrix, preventing further backward movement. In contrast, translocation by cross-bridge ratchet mechanism involves precursor unfolding coupled to an ATP-dependent conformational change of mtHsp70. mtHsp70 generates an ATP-dependent pulling force to transport precursors into the matrix. Accelerated precursor unfolding facilitates its unidirectional forward movement and transport into the matrix.

Mitochondrial protein import is powered by two distinct energy sources: ATP hydrolysis and electrochemical potential across the inner membrane. Newly ...

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JoVE Core

Cell Biology

Unit 1

Intracellular Compartments and Protein Sorting

intracellular-compartments-and-protein-sorting

KEY TERMS AND CONCEPTS

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, polypeptides must unfold. Some precursors with shorter presequences unfold globally on their own, while precursors with longer positively charged presequences undergo local unfolding.

The unstructured presequence then passes through the TOM/TIM pathway. It is helped inward by the negative charges on the inner membrane. Inside the matrix, mitochondrial Hsp70, or mtHsp70, works with TIM44 to recognize the emerging polypeptide and move it fully into the matrix by speeding up unfolding.

mtHsp70 can also trap loosely folded precursors as they enter. In this case, it does not need a conformational change. Tightly folded polypeptides are different. Their translocation triggers ATP-dependent conformational changes in mtHsp70, and ATP hydrolysis helps pull the incoming peptide across the TIM translocase. When ATP binds again, mtHsp70 opens and releases the precursor into the matrix.

Two models explain how mtHsp70 drives precursor movement. In the thermal ratchet model, mtHsp70 uses ATP hydrolysis to bind proteins that unfold on their own and trap them in the matrix so they cannot move backward. In the cross-bridge ratchet model, precursor unfolding is linked to an ATP-dependent shape change in mtHsp70. This creates a pulling force that helps move the precursor forward in one direction.

Mitochondrial Protein Import Energy

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...

translocation of proteins into the mitochondria

translocation-of-proteins-into-the-mitochondria

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...

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 ATP hydrolysis and membrane potential power mitochondrial protein import, from Hsp70 chaperones and TOM/TIM entry to mtHsp70 ratchet models.

Mitochondrial Protein Import Energy | 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 Import Energy

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