Protein Trafficking

Question 1

Where does protein synthesis occur?

Where could the proteins be transferred to?

Answer 1

Nuclear-encoded proteins begin synthesis in the cytosol.

From the cytosol they can be transported to:
nucleus, mitochondria, ER, chloroplast, peroxisomes, etc.

Question 2

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

Answer 2

SORTING SIGNALS

signal sequence OR signal patch

Question 3

What are signal sequences?

Answer 3

exposed, single stretch of amino acids, often at the end of the polypeptide chain (N-terminus)

these can be cleaved/uncleaved after targeting

Question 4

What are signal patches?

Answer 4

amino acids contributing to the signal are separate until after the protein folds

Question 5

List some examples of signal sequences.

Answer 5

NUCLEAR IMPORT
lysine and arginine-rich sequences

MITOCHONDRIAL IMPORT
amphipathic alpha helix

ER IMPORT
hydrophobic amino acids

Question 6

How does a protein (a large macromolecule) cross the lipid bilayer?

Answer 6

PROTEIN TRANSPORT

Proteins have a signal sequence which sticks out at the end. There are receptors that recognise this signal on the membrane which act as a channel through which to guide the protein.

Question 7

Where do all nuclear-encoded proteins begin synthesis?

Answer 7

In the cytosol

Question 8

What are the channels by which proteins enter the NUCLEUS from the cytosol?

Answer 8

NUCLEAR PORES

Nuclear pores are made up of nucleoporins

Small molecules (<5000 daltons) freely diffuse through nuclear pores

Question 9

What state must proteins be in to enter or leave the nucleus?

Answer 9

Proteins transported are post-translational (folded proteins)

Question 10

Describe the mechanism by which proteins are IMPORTED into the NUCLEUS.

Answer 10

CHANNEL — nuclear pore
RECEPTORS — nuclear import receptors
SIGNAL — nuclear localisation signals

NLS on the protein binds to IMPORTIN (nuclear import receptor) which binds to a nucleoporin binding site on the nuclear pore

Question 11

Describe the mechanism by which proteins are EXPORTED into the NUCLEUS.

Answer 11

CHANNEL — nuclear pore
RECEPTORS — nuclear export receptors
SIGNAL — export signal

Export signal on the protein binds to EXPORTIN (nuclear export receptor) which binds to a nucleoporin binding site on the nuclear pore

Question 12

What type of substance are Importin and Exportin?

Answer 12

Karyopherins

Question 13

What is Ran-GTPase? What does it do?

Answer 13

Ran-GTPase is a ???

Question 14

What is meant by translocation?

Answer 14

The transport of a protein across a membrane.

Question 15

What is meant by TOM and TIM?

Answer 15

TOM — Translocon of the Outer Membrane

TIM — Translocon of the Inner Membrane

Question 16

Describe the mechanism by which proteins are IMPORTED into the MITOCHONDRIA.

Answer 16

CHANNEL — TOM and TIM complexes
RECEPTORS — TOM complex

The signal sequence of the post-translational unfolded protein is recognized by a receptor protein in the TOM complex. The protein is then translocated through the TOM complex and transferred to the TIM complex.

Question 17

What are the energy requirements for protein import into the mitochondria?

Answer 17

ATP

Electrochemical gradient across the inner membrane

Question 18

What happens to a protein once it has been imported into the mitochondrial matrix?

Answer 18

The protein is cleaved by SIGNAL PEPTIDASE, producing a mature mitochondrial protein.

Question 19

How is the unfolded protein kept unfolded during translocation?

Answer 19

The protein binds cytosolic Hsp70

Question 20

Define “nascent” in the context of proteins.

Answer 20

A nascent polypeptide chain is one which is newly synthesized

Question 21

Define “glycosylation”

Answer 21

Glycosylation refers to the addition of sugars to a protein or lipid

Question 22

Describe the process of co-translational import of proteins into the endoplasmic reticulum (ER)

Answer 22

CHANNEL — Sec61 complex
RECEPTORS — SRP receptor
SIGNAL — SRP (signal recognition particle)

SRP binds to the signal sequence on the protein, causing translation to pause. The SRP-bound ribosome attaches to the SRP receptor on the ER membrane.

The protein remains attached to the translocon on the ER membrane, translation resumes and translocation begins.

SRP and the SRP receptor are displaced and recycled.

Question 23

What role does GTP hydrolysis play in protein translocation into the ER?

Answer 23

GTP hydrolysis powers assembly of the nascent chain — translocon complex, as well as release of the SRP / SRP receptor

Question 24

What are the two types of protein import into the ER?

Answer 24

Co-translational and Post-translational

Question 25

Describe the process by which naked (uncoated) vesicles dock and fuse with a target membrane.

Answer 25

1. Vesicles contain a protruding v-SNARE and an external Rab-GTP
2. The vesicle is tethered as the Rab-GTP binds to a Rab effector, a tethering protein, and the vesicle is pulled towards the membrane
3. Once close to the membrane and turned to the right configuration, the protruding v-SNARE binds to t-SNAREs on the membrane, forming a SNARE complex
4. The SNARE complexes on either side pinch and fuse the vesicle membrane and the plasma membrane together to allow for the contents of the vesicle to leave the cytosol into the extracellular space
5. The SNARE complexes are then bound and disassembled by alpha-SNAP (an adaptor protein). The ATPase NSF catalyses the ATP-dependent unfolding of the SNARE proteins and releases them into the cytosol for recycling.

Question 26

How does the Golgi modify proteins?

Answer 26

O-linked glycosylation

Sugars are added to -OH groups of serine/threonine

This process is catalysed by a series of glycosyl transferase enzymes in the Golgiq

Question 27

What are the two models for organisation of the Golgi and the transport of proteins between ER and Golgi?

Answer 27

Vesicular Transport Model

Cisternal Maturation Model

Question 28

What is the lysosome?

Answer 28

The lysosome is an organelle which contains proteases and lipases for degrading proteins and lipids.

It fuses directly with the late endosome and is then reformed. Its lumen is acidic.

Question 29

Discuss the use of yeast screens to find genes involved in protein trafficking to the vacuole (i.e. the yeast lysosome).

Answer 29

The screens involved mutagenizing yeast and assaying for secretion of a vacuolar protease.

Over 80 genes have been identified (known as VPS [vacuolar protein sorting] genes)

The VPS genes encode SNAREs, small GTPases (Rabs), tethers, sorting proteins

All VPS genes have homologues in mammalian cells.

Question 30

How was the LDL receptor endocytosis signal discovered?

Answer 30

Goldstein and Brown were studying patients with familial hypercholesterolaemia

One patient had an amino acid change:
-NPVY- to -NPVC-
in the cytoplasmic domain of the LDL receptor

LDL still binds the mutant receptor, however it fails to internalise; LDL cannot be cleared from the blood

Question 31

What does LDL stand for?

Answer 31

Low Density Lipoprotein

i.e. cholesterol

Question 32

What does EGF stand for?

Answer 32

Epidermal Growth Factor

Question 33

How would you study protein trafficking using transfection methods?

Answer 33

To find the protein of interest, tag the protein with GPF, transfect it into the cell, and find it.

Question 34

How can viruses enter the cell?

Answer 34

Endocytosis

e.g. influenza