The need for protein targeting/ trafficking, general principles of protein transport across cellular membranes, nuclear import, mitochondrial import

Question 1

Into what kinds of compartments are cells divided ?

Answer 1

Eukaryotic cells are divided into membrane-bound compartments called organelles ?

Question 2

What is the difference between the cytoplasm and the cytosol ?

Answer 2

Cell = cytoplasm + nucleus
Cytoplasm = cytosol + organelles

Question 3

Between which organelles/compartments can we observe gated transport ?

Answer 3

The cytosol and the nucleus.

Question 4

Between which organelles/compartments can we observe transmembrane transport ?

Answer 4

The cytosol and :

• mitochondria
• endoplasmic reticulum
• plastids
• peroxisomes

Question 5

Between which organelles/compartments can we observe vesicular transport ?

Answer 5

The ER and the golgi
The golgi and : 
- late endosomes
- lysosomes
- early endosomes
- the cell exterior
- secretory vesicles
... etc.

Question 6

Where do nuclear proteins begin synthesis ?

Where can these proteins be transported thereafter ?

Answer 6

• Nuclear-encoded proteins begin synthesis in the cytosol
• From the cytosol they can be transported to:
- nucleus
- mitochondria
- ER
- plastids e.g. chloroplasts, peroxisomes

Question 7

How does a protein know where to go in the cell ?

Answer 7

Through sorting signals :

• signal sequences : exposed, single stretch of AAs often at the end of the polypeptide chain
• signal patch : AAs contributing to the signal are separate until after the protein folds

Question 8

What are signals for :

• nuclear import ?
• mitochondrial import ?
• ER import ?

Answer 8

Nuclear import : –PPKKKRKV– (lysine and arginine-rich sequences)
Mitochondrial import : +H3N-MLSLRQSIRFFKPATRT LCSSRYLL– (amphipathic α helix)
ER import : +H3N-MMSFVSLLLVGILFWATEAEQLTKCEVFQ– (hydrophobic amino acids)

Question 9

Are signals cleaved or uncleaved after targeting ?

Answer 9

Signal sequences can be cleaved or uncleaved after targeting.

Question 10

How does a protein (even with a signal) cross a lipid bilayer ?

Answer 10

• A receptor recognizes signal
• A Channel through which to guide protein
• Receptors and channels are made of proteins

Question 11

What are nuclear localization signals (NLSs) ?

Answer 11

A NLS is an AA sequence that “tags” a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines (K) or arginines (R) exposed on the protein surface.

Question 12

How do proteins enter the nucleus ?

Answer 12

• Proteins enter the nucleus via NUCLEAR PORES •Small molecules (< 5000 daltons) can freely diffuse through nuclear pores
• Larger molecules are imported via active transport

Question 13

What are nucleoporins ?

Answer 13

Nucleoporins = proteins that make up the nuclear pore complex :

• cytosolic fibrils
• annular subunits
• lumenal subunits
• column subunits
• ring subunits
• nuclear fibrils
• nuclear baskets

Question 14

How big are nuclear pores ?

Answer 14

About 0.1um (100nm) wide

Question 15

How does nuclear protein import occur ?

Answer 15

• the folded protein along w/ its NLS is recognized by a nuclear import receptor (karyopherin family) = importin
• nucleoporins have “FG repeats” (Phe + Gly) that serve as binding sites for the import receptors

Question 16

How does nuclear protein export occur ?

Answer 16

• the folded protein along w/ its NLS is recognized by a nuclear import receptor (karyopherin family) = exportin
• nucleoporins have binding sites for the export receptors

Question 17

What is the role of the Ran-GTPase ?

Answer 17

The Ran-GTPase drives directional transport through nuclear pores :
- for nuclear import, Ran-GTP binds the nuclear import receptor in the nucleus whilst the cargo is delivered, and after export, Ran-GTP is hydrolyzed by Ran-GAP (Ran-GTPase Activating Protein) to Ran-GDP + Pi and releases the nuclear import receptor
- for nuclear export, Ran-GTP binds the nuclear export receptor (caring a protein w/ a nuclear export signal) in the nucleus and transports it out of the nucleus, where it is hydrolyzed by by Ran-GAP to Ran-GDP + Pi
Every time Ran-GDP re-enters the cell, Ran-GEF (situated on the chromatin in the nucleus) triggers the exchange of GDP for GTP

Question 18

Is nuclear protein import pre or post-translational ?

Answer 18

Post-translational, proteins are folded before import

Question 19

What is the signal for the transport of protein into the mitochondria (e.g. cytochrome oxidase, subunit IV) ?

Answer 19

An amphipathic α helix : positively charged residues on one side of helix, uncharged residues on the other

Question 20

Mitochondria are double-membrane- enclosed organelles.

Where does translocation occur ?

Answer 20

At special sites where the inner and outer membranes are close together.

Question 21

Are proteins imported in the mitochondria as folded or unfolded polypeptide chains ?

Answer 21

Unlike nuclear import, proteins are imported as unfolded polypeptide chains.

Question 22

How are matrix proteins imported ?

Answer 22

• The signal sequence is recognized by a receptor (part of the translocon of the outer membrane [TOM] complex)
• The unfolded polypeptide is translocated through TOM and transferred to the translocon of the inner membrane [TIM], specifically TIM23
• The protein to be translocated is kept unfolded by the binding of cytosolic Hsp70 (ATP hydrolysis necessary). and as the polypeptide travels through TOM, Hsp70 molecules dissociate)
• Translocation across the inner membrane requires the electrochemical gradient (+ve outside Vs -ve inside)
• The signal sequence is cleaved by a protease and a mitochondrial Hsp70 binds to drive further translocation
• Hsp60 helps the translocated protein to fold correctly

Question 23

How are proteins inserted into the outer mitochondrial membranes ?

Answer 23

• The polypeptide is translocated through TOM, kept unfolded in the intermembrane space by cheprones
• The polypeptide is then inserted by the SAM (Sorting and Assembly Machinery) complex into the outer membrane, where it fully folds

Question 24

How are proteins transported to :

• the inner membrane (anchored) ?
• the inter-membrane space ?
• the inner membrane (integral) ?

Answer 24

Inner membrane (anchored) :
–> form the cytosol :
- polypeptide in translocated through TOM and TIM
- when the stop-transfer (hydrophobic region) reaches the inner membrane, the signal sequences (now inside the matrix) is cleaved
–> from the matrix (or form the cytosol and already cleaved but possesses a second signal) :
- polypeptide is integrated in the inner membrane via the OXA complex
Inter-membrane space :
- when strop transfer sequence reaches the inner membrane, the polypeptide is cleaved and free in the inter-membrane space
Inner membrane (intergal) :
- polypeptide translocated through TOM
- kept unfolded by inter-membrane space chaperones
- integrated into the inner membrane by the TIM 22 complex (requires electrochemical gradient)