Question

Question 2

1. How is a protein targeted to the matrix of mitochondria in a eukaryotic cell?
2. Describe the “travels” of a newly transcribed polypeptide as it goes from ribosome to its destination in the matrix.

(Hint: the words “contact site” should occur somewhere in this description if your polypeptide is traveling the right path.)

Answer 1

Protein synthesis, irrespective of their final destination, always initiates in the free ribosomes of cytoplasmic compartment of the cell. Further fate is defined by the targeting signals within the sequence of amino acids in the proteins. Hence each protein is predestined on account of its amino acid sequence to perform different functions and different proteins contain different addressing information that enables them to be routed to different specific destinations. Any change or mutation within this sequence results in improper transport and processing of preoteins.

There are three major types of transport systems, viz.

a. Nuclear pores

b. Translocation across membranes.

c. Vesicle transport

Major problem being transportation of largely hydrophilic proteins across the hydrophobic barrier presented by the membrane core.However,  it is resolved in the same way that transporters for smaller hydrophilic molecules work, by creating a controlled hydrophilic passage through the membrane for specific molecules. Basically, signal information is the key factor. It corresponds to the address on a letter and is contained in the amino acid sequence of the protein. It allows the protein to be recognized and processed by other proteins.

In general, the signal sequences are located at or near the amino-terminal end of the polypeptide. Each of the signal sequences has a characteristic amino acid motif and are critical for protein targeting.

Broadly, protein targeting to mitochondria comprises; recognition of  protein containing signal sequence, binding, unfolding on entry and refolding on the organellar side of the membrane.This step requires a set of special organellar chaperone proteins. The protein recognition signal is quite large. The entry site to mitochondria is a site where the inner and outer membranes touch. This is referred to as a contact site. Thus ;

1.The protein containing the signal sequence is synthesized in the cytoplasm.

2.Signal sequence binds to a receptor in the organelle membrane

3. Receptor - protein complex diffuses within membrane to a contact site.

4. Protein is unfolded, moved across the membrane, and refolded. These operations are carried out by the protein transporter complex and its associated chaperone proteins.The signal sequence is the first part of the protein to enter the organelle.

5. Once inside, the signal sequence is cleaved off by a specific peptidase.

The outer membrane of the mitochondria contains the protein "porin". This forms an aqueous channel through which proteins up to 10,000 daltons can pass and go into the intermembrane space. Indeed, the small molecules actually equilibrate between the outer membrane and the cytosol. However, most proteins cannot get into the matrix unless they pass through the inner membrane. This membrane contains cardiolipin which renders it virtually impermeable. This requires transport mechanisms across the membrane that are more organized and regulated. Transport across the mitochondrial membranes requires the concerted action of a number of translocation machineries. The machinery in the outer membrane is called the Tom complex (Translocator outer membrane) and that for the inner membrane is called the Tim complex (Translocator Inner Membrane). Proteins that have to go all the way to the matrix have an NH₂ cleavable signal sequence. Most proteins must be uncoiled or stretched out to go through the translocators. This involves ATP binding and is monitored and stabilized by a chaperone proteins, including hsp70. Thus, before the protein can go through Tom complex, it must become "translocation competent".

Not surprisingly, the TOM complex will include import receptors that initially recognize the signal peptide or a signal sequence (these include Tom20, Tom22, and Tom70). Different proteins use different receptors.The receptors then bring the protein to the region containing the translocator proteins. This is actually a complex of proteins.  It is called the General Import Pore (GIP) and it facilitates the translocation of the presequence of the protein across the outer membrane. (the GIP is made of Tom40, Tom5, Tom 6, and Tom7). Tom40 appears to be the core element of the pore and forms oligomers. It traverses the membrane as a series of 14 anti-parallel beta strands which form a beta barrel. It also interacts with polypeptide chains passing through the pore. All of the other Tom components in GIP are anchored to the outer membrane by helical transmembrane segments (hydrophobic anchors).  

Mitochondrial proteins destined for the matrix often have a cleavable signal peptide on the protein which must be recognized before it will be admitted through the mitochondrial translocator. These proteins with "amino terminal signals" (your text), or "preproteins" or "presequences" (current literature) usually interact with Tom20 first. Then, they have to get through the outer membrane. To do that, they are transferred to the GIP complex: First, they interact with Tom22 and Tom5 which ushers them to the pore formed by Tom40. They then enter the matrix using the pore complex made of Tim23 and Tim17 which are in the inner membrane. Also, very important, their entry is dependent on membrane potential. This is set up by the electron transport complexes. Recall that hydrogen ions are being pumped into the intermembrane space creating a charge gradient that is more negative on the matrix site. This membrane potential actually helps pulls the protein into the Tim23-Tim17 channels. The protein then enters the matrix where the cleavable preprotein is clipped off by a protease, MPP. mt-hsp 70 in the matrix works with Tim44 to complete the full transfer to the matrix. mthsp70 and Tim 44 actually "pull" the protein into the matrix by a process that requires ATP. It also requires the membrane potential set up by the electron transport chain.

Some mitochondrial proteins destined for the inner membrane have a cleavable presequence followed by one or more hydrophobic membrane-spanning segments that function as stop-transfer sequences in the IM or, serve to insert the polypeptide into the IM after it gets in the matrix. These are like the Type I membrane proteins described in the unit on the rough endoplasmic reticulum. However, other proteins do not have a cleavable targeting signal (Types II and III). Mitochondrial proteins that have an internal signal sequence (examples include a number of proteins in the inner membrane) generally interact with Tom70 as the receptor. Then, after they transit the outer membrane via the GIP complex, they enter the special Tim pathway. This may involve interactions with small Tim's of the intermembrane space and Tim22-Tim54 of the inner membrane itself.

Those proteins that do not have a cleavable targeting signal sequence often have signals with the following characteristics: They are often a stretch of positively charged amino acids (sometimes adjacent to a membrane spanning hydrophobic region). Sometimes these form loops that face the matrix. Recall the "positive inside rule" has positively charged amino acids concentrated at the cytosolic side for proteins being inserted into the rough endoplasmic reticulum. These mitochondrial proteins tend to follow this rule, only the matrix becomes the site where the positive charges are most numerous.

Entire sequence of events required to take a protein into the matrix is represented in the following illustration

1. Protein unfolds as it binds to hsp70 chaperone. Red positive area indicates targeting sequence. Chaperone binding is ATP dependent.

2. Targeting sequence binds to receptor (usually Tom20)

3. Receptor ushers protein to site of translocator. Other Tom proteins involved, but Tom40 is the core of the translocator channel.

4. Protein is translocated stimulated by the membrane potential. Electron transport complexes on inner membrane have pumped H+ across to the intermembrane space, leaving the matrix more negative. This attracts the protein (the signal is positively charged). Protein moves through Tim translocators. Tim 44 and hsp70 in the matrix continue to guide and pull the protein through the pore. An ATP requiring process.

5. another chaperone (called a chaperonin), hsp60 causes the folding of the the protein into its tertiary sequence. Also an ATP requiring process.

6. Presequence is cleaved in the matrix.