Protein Sorting (Ch.15)

Peroxisomes contain. . .

1+ Enzymes that produce Hydrogen Peroxide

The signal sequence is both necessary and sufficient to. . .

Direct the protein to its destination

Once inserted into a membrane a transmembrane protein. . .

Does not change its orientation

How does the overall process of importing a protein into the nucleus work then?

1. A nuclear protein with a nuclear localization signal makes itself present. 2. The nuclear protein binds to the NIR and is delivered to the nuclear pore. 3. Ran-GTP binds to the receptor and provides it with the energy to move through the pore. 4. Ran hydrolyzes it bound GTP and Ran-GDP disassociates from the receptor

Start-signal sequences (NOT THE SAME THING AS N-TERMINUS) are typically not removed from polypeptide polypeptides that are embedded into the membrane. What does this create?

A double pass (multi-pass) transmembrane protein.The polypeptide chain passes back and forth across the lipid bilayer. Hydrophobic signal sequences are thought to work in pairs; they have an internal start transfer sequence that serves to initiate translocation until a stop-transfer sequence is reached. Both hydrophobic sequences are released into the bilayer where they remain as membrane-spanning alpha helices. They have both their N-Terminus and C-terminus on the lumen side and they have more loops.

What specific signal does the protein display?

A nuclear localization sequence (NLS)

What is a single-pass transmembrane protein?

A protein inserted in the membrane with a defined orientation; these only have one hydrophobic region spanning the membrane (N-terminus on the lumen side of the bilayer and the C-terminus on the cytosolc side)

What is the difference between a start signal sequence and an n-terminus?

A start signal sequence is a specific point on the polypeptide that tells translocation where to begin. An N-terminus is the exact point where the signal first starts at.

The translocation process for proteins that remain embedded to the ER membrane is a lot more complicated (THESE ARE NOT SOLUBLE PROTEINS). Explain the process.

A transmembrane protein with a single membrane-spanning segment has a N-Terminus that initiates translocation (similar to soluble proteins; can also be a start transfer sequence if it is not an N-terminus). However, a STOP-TRANSFER sequence (an additional sequence of hydrophobic amino acids) halts the transfer process further along the polypeptide chain. The translocation channel releases the growing protein SIDEWAYS into the lipid bilayer. N-terminus is cleaved off, while the sto-transfer sequence remains in the bilayer.

Soluble proteins made on the ER are released into the. . .

ER lumen

What is a sorting signal?

Amino acid sequences found in transported proteins that selectively guide the distribution of the proteins to specific cellular compartments

Once again, what is a sorting sequence?

An active, short stretch of the amino-acid sequence

GTP hydrolysis drives nuclear transport in the. . .

Appropriate direction

What is a SRP?

Certain particles in the cytoplasm that bind to ribosomes and ER signal sequences

If you remove a signal sequence from an ER protein it becomes a. . .

Cystolic protein

The import of material into the nucleus requires. . .

Energy

What is another difference between Double pass and single pass?

For membrane proteins, single pass means that the polypeptide chain goes through the membrane once. Double pass means that the polypeptide chain goes through the membrane twice. Therefore, a single pass membrane protein has its C terminus and its N terminus on opposite sides of the membrane. A double pass membrane protein has its N terminus and C terminus on the same side of the membrane (since the protein has to loop to go through the membrane a second time.

Where do Proteins enter the Peroxisomes?

From Both the Cytosol and ER

How do peroxisomes get their proteins?

From selective transport from the cytosol (a short sequence of three amino-acids act as import-signal for the peroxisome proteins. They are recognized by receptor proteins in the cytosol.

NIRs help a new protein. . .

Get to a nuclear pore by interacting with the fibrils on the cystolic side of its pore

The inner nuclear membrane is responsible for. . .

Having binding sites for chromosomes

What exactly is a start/stop transfer signal?

Hydrophobic stretches of amino acids which favorably interact with the hydrophobic interior of phospholipid membranes

How can you tell if a signal works?

If you take the sequence of A-A and attach it to proteins that would typically not be in that place, yet the proteins still switch places they are supposed to

The protein is delivered in which conformation?

It 3D Shape

What does the smooth ER do?

It facilitates the creation of lipids such as steroids

When the stop-transfer sequence remains in the bilayer, what structure does it have?

It is a alpha helical membrane spanning segment that anchors the protein in the membrane.

What is the composition of a nuclear pore?

It is composed of 30+ different proteins; these proteins have unstructured, regions that act as a mesh. It has fibrils sticking out of the cytosolic side of the nucleus, and it has a nuclear basket sticking into the nuclear side.

What does the rough ER do?

It is the site of protein synthesis and it constitutes for the majority of the ER

The Endoplasmic Reticulum is an example of a membrane enclosed organelle. What does it do?

It is the site of synthesis of new membranes (e.g large parts of ER have ribosomes attached a.k.a the rough ER)

If a protein has a sorting signal. . .

It moves from the cytosol to the organelle

What must a newly made protein have to ensure that it can actually move into the nucleus?

It must display the proper sorting signal (specifically, it must have a signal sequence that tells the protein to move from the cytoplasm into the nucleus)

What does the end of the signal sequence do?

It opens up the channel on the protein translocator

The Golgi Apparatus is another example of a membrane bound organelle. What does it do?

It receives proteins and lipids from the Endoplasmic Reticulum; it modifies them and sends them to other parts

If a protein lacks a sorting signal. . .

It will stay in the cytosol

What will happen to a folded protein as it begins to request entrance into a mitochondria and/or chloroplast?

It will unfold

What is the importance of the NLS?

NLS are the specific signals that proteins must be able to display so they can be recognized by the nucleus

Certain larger molecules and macromolecules need to pass through the pores such as. . .

Newly made proteins that must be imported from the nucleus from the cytosol

How does the nucleus communicate with the environment?

Nuclear Pores

What specific nucleus signal recognizes the protein's NLS to allow it to actually get into nucelus?

Nuclear import receptors (NIR)

The nuclear envelope is perforated by. . .

Nuclear pores

The nuclear envelope encloses the. . .

Nucleus' DNA and defines a nuclear compartment

What happens when the SRP is done?

Once bound to the ribosome, the SRP is released. The receptor passes the ribosome to a protein translator in the ER membrane and protein synthesis recommences (the polypeptide is threaded across membranes through channels)

What ultimately causes a soluble protein to be released into the lumen?

Once it released from the channel, the c-terminus must pass through the translocaton channel

What is the third way that proteins tend to move across membranes?

Proteins from the ER tend to move through transport vesicles and are transported into one compartment of a endomembrane system (the transport vesicle is pinched off one the membrane of 1 compartment and it fuses with the membrane)

Why do proteins unfold as they attempt to enter the mito or chloro?

Proteins have a signal sequence at the n-terminus that allows them to enter their specific organelles. Proteins that are destined for another organelle are translocated simultaneously.

What typically happens to the sorting sequence is often. . .

Removed from the finished protein post-sorting

The outer nuclear membrane is responsible for. . .

Resmebling and continuing the ER

Proteins are transported into organelles by three mechanisms. What are all these proteins made by?

Ribosomes

Proteins components that help ER signals sequences move to the ER Membrane include. . .

Signal Recognition Particles (SRP) and SRP Receptors

What is a SRP receptor?

Signals embedded in the ER membrane that recognizes SRP

What is the main difference between single-pass and double-pass (multi-pass) membrane proteins?

Single pass proteins have an n-terminal signal sequence that is cleaved off, while the stop transfer sequence remains in the bilayer. Double pass proteins have an internal signal sequence that it used to start the protein transfer (start-transfer sequence) that is never removed from the polypeptide. A protein with two transmembrane domains would have at minimum a start-transfer and a stop-transfer sequence. The first one would lead to the anchoring of one domain in the membrane and the second one would anchor the second transmembrane domain. Double passes have at most a start transfer and stop transfer where the first one anchors it to the bilayer and the second anchors the second transmembrane domain (For membrane proteins, single pass means that the polypeptide chain goes through the membrane once. Double pass means that the polypeptide chain goes through the membrane twice. Therefore, a single pass membrane protein has its C terminus and its N terminus on opposite sides of the membrane. A double pass membrane protein has its N terminus and C terminus on the same side of the membrane (since the protein has to loop to go through the membrane a second time).

The binding of the SRP to the ribosome that shows the ER signal sequences. . .

Slows down protein synthesis

What causes differentiation in destinations?

Specific sequences can vary greatly because of hydrophobic interactions of the charged amino acids

How do Proteins destined for other MEOs move through organelles?

Transport vesicles bring individual proteins from organelle to organelle or to the plasma membrane

Each protein is. . .

Unfolded as it is transported and the signal sequence is removed post translocation

Multipass proteins are. . .

Stitched into the lipid bilayer as they are being synthesized because they use multiple pairs of start and stop transfer sequences, where one reinitiates translocation and the other stops it and causes polypeptide release

Where do all Proteins destined to go into other MEOs enter first?

The Endoplasmic Reticulum

The end of the signal that is made first tends to be. . .

The N-Terminus

What does the fate of the protein depend on?

The amino-acid sequence which has a sorting signal that directs the protein to the specific organelle it's supposed to go to

Most membrane bound organelles are held in location by what?

The attachment to cytoskeleton, especially to microtubulues

What are membrane enclosed organelles surrounded by?

The cytosol and enclosed by a plasma membrane (e.g nucleus is surrounded by a double membrane defined by a nuclear envelope)

What is the second story about the evolution of MEOs?

The endosymbiotic theory (mitochondria and chloroplasts co-evolved independently against each other)

Where does the nucleus get this energy to import molecules into it?

The hydrolysis of GTP by a GTPase called Ran

What is the second way that proteins tend to move across membranes?

The proteins move from the cytosol into ER, Mitochondria or Chloro

What is the process for the opening of the channel of the protein translocator?

The signal sequence remains bound to the channel while the rest of the chain is threaded through the membrane as a large polypeptide chain. It is then removed by a transmembrane signal peptidase, which has an active site facing the lumen side of the ER membrane. They are, then, released from the channel into the lipid bilayer and degraded.

What type of proteins go from the cytoplasm to the ER?

Water soluble proteins that translocated from the ER membrane into the lumen or perspective transmembrane proteins that are partly translocated across ER membrane to become embedded

What is the job of a nuclear pore?

They allow molecules to leave or enter (similar to gates)

What are protein translocators?

They facilitate movement of proteins across membranes

How do the NIRs actually penetrate the pore to receive the signal?

They penetrate the pore with "stabby" short Amino-Acid sequences within the mesh of its nuclear basket

What is the first way that proteins move across membranes, which are typically extremely hydrophobic regions (nucleus)?

They tend to move through nuclear pores, penetrating the inner and outer; this is an example of active transport

MEOs have evolved in different ways. What is the first story?

They were thought to be microorganisms with no plasma membrane but internal organelles. The first story was thought to be an invagination of the plasma membrane

Where do proteins tend to enter the nucleus?

Through nuclear pores

What aids in the transport of the protein into the peroxisome?

Translocators in the peroxisomal membrane

Proteins are able to enter the ER while. . .

being made!