Protein trafficking: ER and mito

Question 1

Describe the basic principles of protein sorting in the cell.

Answer 1

1. information to target protein to a particular organelle is encoded in the amino acid sequence of the protein
2. each organelle carries a set of receptors that bind to specific signal sequences (e.g. SRP receptor in ER)
3. unidirectional transfer is often achieved through coupling transfer to an energetically favorable process such as ATP hydrolysis

Question 2

Why would evolution stop without mito?

Answer 2

Glycolysis harnesses only 10% of the power of ATP

Question 3

What does it tell us about the origin of mito that they are surrounded by two membranes?

Answer 3

That they came from invagination of bacteria

Question 4

Matrix vs intermembrane space of mito

Answer 4

Matrix is at very center, intermembrane space is between the two membranes

Question 5

Where are mito proteins synthesized?

Answer 5

Fully synthesized in cytoplasm before being translocated into mito

Question 6

Describe signal sequence for proteins destined to the mito.

Answer 6

proteins destined for matrix space have Nterminal signal sequence:

• amphipathic alpha helix with hydrophobic residues clustered on the opposite side
• alternating positive, nonpolar, and hydrophobic residues to get hydrophobic residues all clustered on the same side in secondary structure
• the structure gets the protein translocated, not the primary sequence

Question 7

List the principle protein translocators needed to import proteins into mito.

Answer 7

each is a multiprotien complex:

• TOM (translocator of the outer mito membrane)
• TIM23
• TIM22
• SAM
• OXA

Question 8

What is the role of the TOM complex in transport of proteins to mito?

Answer 8

Used for translocation of all proteins encoded by the nucleus. Consists of receptor and translocation channel. It is on the outer mito membrane and recognizes the unfolded protein’s signal sequence structure. Transports proteins from the cytosol to the intermembrane space through translocation channel, as the Hsp70 chaperones that kept the protein unfolded are shed.

Question 9

Describe the TIM23 complex.

Answer 9

Spans inner to outer membrane right near TOM. Further transports proteins into the matrix space after TOM moves them into intermembrane space (requires ATP). After signal sequence is cleaved in matrix by signal peptidase, the protein folds properly.

Also helps to insert some inner membrane proteins.

Question 10

Describe the TIM22 complex.

Answer 10

Needed for the insertion of a different subset of inner membrane proteins

Question 11

Describe the SAM complex.

Answer 11

Helps to fold proteins properly within the outer mito membrane

Question 12

Describe the OXA complex.

Answer 12

involved in inserting proteins synthesized within the matrix into the inner membrane of the mito.

Question 13

What experiments can we do to know mitochondrial membrane transport takes place?

Answer 13

1. incubate labelled protein with mito and centrifuge. If the protein is transported into the mito, they will fractionate together in the centrifuge tube.
2. Add protease to degrade signal sequence if it has not been imported into the mito. (removal of sequence can be monitored using gel – if removed, it will move further)
3. If signal is not removed, we know it has been imported. Can confirm by adding detergent to disrupt membrane of mito and then add protease.

Question 14

How can we use varying temperatures to study import of proteins into the mito?

Answer 14

1. expose mito to proteins on ice (nothing will happen, too cold, or will occur slowly. Add protease, and might only find signal sequence inside mito, showing that it is imported N-term first)
2. if you warm up the reaction and allow it to proceed, we will find the whole protein inside the mitochondria even after protease is found because the mito protects the protein from degradation
3. can then add detergent to break down mito membrane and recover protein

Question 15

What steps of mito protein import require energy?

Answer 15

1. release of unfolded proteins in the cytosol from Hsp70 chaperones after binding to TOM complex requires ATP
2. translocation of protein through TIM23 channel relies on proton gradient (higher in intermembrane space than matrix)
3. mitochondrial Hsp70 helps pull unfolded protein into matrix space and requires ATP hydrolysis to be released from chaperone

Question 16

Describe process of outer mito membrane insertion of a protein.

Answer 16

• some outer membrane proteins can be directly inserted by TOM
• a large group of outer membrane proteins called porins are initially imported through TOM, but need help folding all of the way (have beta barrel structure)
• in the intermembrane space, these proteins associate with chaperones for folding
• SAM complex in outer membrane then helps these porin proteins properly fold within the outer membrane

Question 17

Describe the process of protein insertion into the mitochondrial inner membrane using just TOM and TIM23.

Answer 17

• TOM and TIM23 complexes allow N-term to enter the matrix space
• hydrophobic sequence within the protein stops further translocation into the matrix and leaves protein in inner membrane
• TOM translocator then pulls the rest of the protein from tbe cytosol to the intermembrane space
• N-terminal sequene of protein within matrix is cleaved, and the protein dissociates from TIM23, remaining anchored in the inner membrane

Question 18

How can we test which mito membrane a protein gets embedded into?

Answer 18

can do site-directed mutagenesis of the protein sequence to see if it changes where it ends up, can then determine the sequence which determines where a protein is embedded.

Question 19

Describe the process of protein embedding into mitochondrial inner membrane involving OXA.

Answer 19

• TOM and TIM23 translocate protein into matrix
• signal peptide (N-term) is cleaved
• hydrophobic sequence is then exposed on N-term, and is recognized by OXA complex, which mediates the insertion of the protein into the inner membrane
• this process involving OXA is used to mediate insertion of inner membrane proteins synthesized within the mito.

Question 20

Describe transport of protein to mito inner membrane using TIM22 complex.

Answer 20

• TIM22 mediates insertion of subclass of inner membrane proteins including carriers of ATP and ADP and phosphate
• these carrier proteins have internal signal sequences instread of N-terminal sequences
• these carriers pass through the outer membrane via TOM complex to intermembrane space, where they are chaperoned to the TIM22 complex for insertion into the inner membrane when TIM22 reaches STOP sequence of carrier protein.

Question 21

Which membrane structure is the ER membrane continuous with?

Answer 21

The nuclear membrane

Question 22

Describe the four main functions of the ER.

Answer 22

1. production of transmembrane proteins and lipids for cell’s organelles
2. makes lipids for mito and peroxisomal membranes
3. almost all proteins secreted to the cell exterior are initially delivered to the ER
4. serves as intracellular Ca2+ store

Question 23

What are transitional ER?

Answer 23

areas of smooth ER from which transport vesicles bud of for transport to the golgi

Question 24

In what cell types are smooth ER prominant?

Answer 24

Lipid-metabolizing cells such as hepatocytes

Question 25

Describe the smooth ER

Answer 25

- no ribosomes - has enzymes that make cholesterol - has enzymes that make lipid components of particles - contains enzymes needed to detoxify lipid soluble drugs and other harmful compounds produced by metabolism - sequesters Ca2+ from the cytoplasm (these regions of smooth ER are called sarcoplasmic reticulum in muscle cells)

Question 26

What are microsomes, and smooth and rough microsomes?

Answer 26

Microsomes are small closed vesicles maintaining ER function that are formed when cells or tissues are homogenized, breaking the ER into fragments (obtained by centrifugation) - rough microsomes are derived from rough ER and are distinguished by density (due to attachment of ribosomes) - smooth microsomes can come from many places and therefore it is hard to tell their origin

Question 27

How can we tell whether a protein is associated with smooth or rough ER?

Answer 27

Can form microsomes with centrifugation and can distinguish the two types by density.

Question 28

Which ER (smooth or rough) does protein production take place at?

Answer 28

Rough ER

Question 29

Describe the ER signal sequence binding flexibility.

Answer 29

- signal sequence binding site is large and hydrophobic and lined by methionines, allowing for differences in signal size and shape - SRP wraps around large ribosomal subunit and binds to ER signal sequence - SRP also binds to elongation factor binding site between large and small ribosomal subunits to halt protein synthesis until ribosome binds ER membrane so premature protein folding does not take place

Question 30

What is a polyribosome?

Answer 30

Many ribosomes coming together along a transcript

Question 31

What is the Sec61 complex and how does it funciton?

Answer 31

- core of the ER transclocon - 3 subunits, with alpha helices surrounding central pore - beta subunit plugs center pore so Ca2+ cannot leak out - pore can open along a seam on its side to let a protein being synthesized come through - bound ribosome forms tight seal so no molecules escape ER - in eukaryotes, four Sec61 complexes form a large translocator assembly

Question 32

In which directions are the ER translocator gated?

Answer 32

In two directions: laterally (important for integration of protein into membrane) and downwards (beta plug, moving proteins into the ER lumen)

Question 33

Describe translocation of a transmembrane protein with an internal signal sequence

Answer 33

\*some proteins have internal signal sequence instead of N-terminal signal sequence - SRP can bind internal signal sequence and bring the ribosome making the protein to the ER membrane - this site serves as a start-transfer signal to initiate translocation - signal sequence remains in the membrane as a single membrane spaning alpha-helix

Question 34

What determines whether the N-term or C-term of a transmembrane protein with internal signal sequence ends up on cytosol or lumen side of the ER?

Answer 34

- more positive amino acid residue before hydrophobic core signal sequence, the N-term will be cytosol side and C-term on lumen side - more positive amino acid residue after hydrophobic core, the N-term will be on lumen side and the C-term will be on cytosol side

Question 35

What is protein disulfide isomerase?

Answer 35

It is an important ER-resident protein that forms disulfide bonds (covalent modification)

Question 36

What is chaperone protein BIP?

Answer 36

BIP is an ER-resident protein that pulls post-translational proteins into the ER through a translocator. - recognizes misfolded and partially folded proteins - aids in protein folding - BIP binds the protein and through ATP-hydrolysis undergoes cycles of binding and release to drive unidirectional translocation.

Question 37

What results from a Sec61 mutation?

Answer 37

Because it is necessary to move proteins into the ER, Sec61 mutations are lethal.

Question 38

How can we detect the translocation of proteins to a given organelle?

Answer 38

- label known organelle protein - label protein of interest (GFP or antibody if we don't want to have GFP disrupting the protein function) - merge images

Question 39

How can we determine the sequence responsible for translocating a protein to a certain organelle?

Answer 39

We can fuse a sequence of interest with a cytosolic protein (sequence will contain HA or MYC tag) and then probe different organelles for the antibody.