Protein Targeting into the Endoplasmic Reticulum (ER)

Sascha Martens, Dr. Bohrgasse 9/5, Room 5.113, sascha.martens@univie.ac.at

Protein Import into the ER

Overview of Protein Trafficking

Proteins are targeted to various cellular locations:

Cytosol
Nucleus
Mitochondria
Peroxisomes
Plastids
Endoplasmic Reticulum (ER)
Golgi
Late Endosome
Secretory Vesicles
Lysosome
Early Endosome
Cell Exterior

Types of Transport

Gated transport: Movement between the nucleus and the cytosol.
Transmembrane transport: Movement across membranes (e.g., ER, mitochondria).
Vesicular transport: Movement via vesicles (e.g., Golgi, endosomes).

The Endoplasmic Reticulum (ER)

ER Structure and Dynamics

The ER is a highly dynamic membrane structure.
It is continuous with the nuclear envelope.
It extends throughout the entire cell.

ER Expansion

The ER can massively expand in cells specialized for secretion, such as plasma cells.
Plasma cells are specialized in the massive secretion of antibodies.

Rough ER

The rough ER is studded with ribosomes.
It is composed of sheet-like structures.

Smooth ER

The smooth ER is composed of tubular structures.
It lacks associated ribosomes.

3D Reconstruction of the ER

The ER is a connected structure composed of sheets and tubules.

Protein Translocation into the ER

Biochemical Assays

Most details about the mechanism of protein translocation into the ER were uncovered in biochemical assays using purified components.

Rough Microsomes (RMR)

Rough microsomes (RMR) are derived from a salt extract containing ribosomes, mRNA, pre-light chain, and light chain.
Components in a salt extract were needed for translocation.
The translocated form of the protein is larger than the cytoplasmic form.

Signal Recognition Particle (SRP)

What was missing from the salt-washed membranes was the Signal Recognition Particle (SRP).
SRP is composed of 6 protein subunits and an RNA called 7S.
Thus, SRP is a ribonucleoparticle.

SRP Function

SRP recognizes the N-terminal signal sequence, binds to it, and halts translation.
This allows SRP to guide the ribosomes to the ER membrane.

SRP Cycle

SRP recognizes the N-terminal signal sequence, binds to it, and halts translation.
This allows SRP to bind the SRP receptor at the ER membrane.
Both SRP and SRP receptor are GTP-binding proteins and interact only when they are GTP-bound.
After binding to each other, they hydrolyze GTP and dissociate.

Ribosome Cycles

mRNA encoding a cytosolic protein remains free in the cytosol (FREE RIBOSOME CYCLE).
mRNA encoding a protein targeted to ER remains membrane-bound (MEMBRANE-BOUND RIBOSOME CYCLE).
There's a common pool of ribosomal subunits in the cytosol.

Genetic Approach to Identify the Translocation Apparatus

Experiment Setup

Wild-type yeast cell: An enzyme is targeted to the ER; the cell dies without histidine as a nutrient.
Mutant engineered cell: Not all of the enzyme is taken up into the ER; the cell lives without histidine as a nutrient.

Temperature-Sensitive Mutants

The cell containing the temperature-sensitive mutant will grow at room temperature.
At a higher temperature (37^{\circ}C), the cells will die because the mutant protein completely fails to function.

Process

Transformation of recombinant DNA molecules into temperature-sensitive yeast cells.
Isolation of yeast cells that can grow at high temperature.
Isolation and sequencing of the recombinant DNA.
This approach identified Sec61, which is the gene encoding the translocon in the ER membrane.

Biochemical Approaches to Study the Translocation Mechanism

Crosslinking experiments:
- Use mRNA lacking a stop codon.
- Use a photo-activatable amino acid.
- UV irradiate and identify proteins using SDS-PAGE.

The Translocation Apparatus: SEC61 Complex

The SEC61 complex forms the translocon channel in the ER membrane.
It consists of α, β, and γ subunits.
A plug prevents small molecules from leaking out of or into the ER when the channel is not engaged in translocation.
During translocation, the unfolded polypeptide chain fills the channel and prevents leakage.

Initial Stages of Protein Translocation

Binding of SRP to the signal peptide causes a pause in translation.
The SRP-bound ribosome attaches to the SRP receptor in the ER membrane.\n3. SRP and SRP receptor are displaced and recycled.
Translation continues, and translocation begins.

Structure of the Mammalian Ribosome-Sec61 Complex

The ribosome interacts with the Sec61 translocon during protein translocation.
The 60S subunit, A/P hybrid state tRNA, 40S subunit, and nascent peptide are all part of the complex.

Translocation Through the SEC61 Channel

Proteins move through the SEC61 channel into the ER lumen.

Translocation of a Soluble Protein

The signal peptide is cleaved by a transmembrane peptidase – the signal peptidase.

Translocation of Membrane Proteins

Single Spanning Membrane Protein - Type I Topology

A stop-transfer sequence halts translocation and anchors the protein in the membrane.
The N-terminus is in the ER lumen, and the C-terminus is in the cytosol.

Single Spanning Membrane Protein - Type II Topology

The N-terminus is in the cytosol, and the C-terminus is in the ER lumen.

Dual Spanning Membrane Protein

Contains both a start-transfer and a stop-transfer sequence.

Multi Spanning Membrane Protein

Contains multiple start-transfer and stop-transfer sequences.
Hydrophilic and hydrophobic regions alternate, creating transmembrane domains.

Tail-Anchored Proteins

Tail-anchored proteins have a C-terminal transmembrane domain.
They are inserted into the ER membrane post-translationally by the Get complex.

SND Pathway

The SND (SRP-independent targeting) pathway for targeting of transmembrane proteins to the ER.
The transmembrane domain (TMD) signal for ER targeting by the SND pathway can be located throughout the sequence.
It acts alongside the SRP and Get pathways to insert membrane proteins into the ER.

Posttranslational Protein Targeting

Prokaryotes

SecY, SecA, ATP, and ADP are involved.

Eukaryotes

Sec62, 63, 71, 72 complex, Sec61 complex, BiP, ATP, and ADP are involved.

Protein Modifications in the ER

GPI Anchor

Some proteins are modified with a C-terminal GPI anchor within the ER.

Glycosylation

Proteins are glycosylated at N-X-S/T sequences while they are translocated.
N-linked glycosyl-groups are initially assembled on the cytoplasmic site of the ER and then transferred en bloc to certain asparagine residues of the protein.

Impact of Glycosylation

Glycosylation has a major impact on the structure of the protein.

The Glycosylation Paradox

Some proteins need to be glycosylated to fold properly, but the location of glycosylation on the protein did not seem to matter.
Glucose moieties were first removed from the proteins and later put on again.
This cycle helps the protein to fold by allowing misfolded protein to interact with chaperone proteins.
Glycosyl-transferase binds to hydrophobic patches on the surface of misfolded proteins.

Protein Folding

Spontaneous Protein Folding

The 3D information for a protein is contained within the primary sequence.
In vitro, it takes several hours or days for protein to fold.
In vivo, it takes only minutes.
5-10% of all E. coli proteins are folding at any one time.
These folding proteins are dangerous for the cell because they are prone to aggregation.
At concentrations found in cells, proteins would completely aggregate in vitro.

Chaperones

Cells have specialized proteins called chaperones that help proteins to fold.

Examples of Chaperones

Calnexin: Folding protein carbohydrate, ERp57, Protein-disulfite isomerase (PDI)
DnaK: Related to the ER chaperone BiP, bound to a peptide substrate
DegP: A cage-forming chaperone found in the bacterial inner membrane space
GroEL-GroES: A cage-forming chaperone found in the bacterial cytoplasm

Unfolded Protein Response (UPR)

ER Stress

What happens if unfolded proteins accumulate in the ER (ER stress)?

Mechanism

Unfolded proteins are bound by a sensor called IRE1.
After binding to these unfolded proteins, IRE1 dimerizes and possibly oligomerizes.
Oligomerization activates a ribonuclease domain that cleaves out an intron from a mRNA encoding the transcription factor XBP1.
After the intron is removed, the mRNA is re-ligated by tRNA ligase, allowing the translation of the XBP1 transcription factor.
In the nucleus, XBP1 activates the transcription of genes that help the unfolded proteins to fold and to expand the ER.

Oligomerization of IRE1

Observed by fluorescence microscopy.

Alternative Pathways to Relieve ER Stress

ATF6
PERK

Protein Degradation

When proteins fail to fold even after several cycles of binding and unbinding to ER-resident chaperones, they are degraded.
This occurs after they have been retro-translocated into the cytosol.

Proteasome

The degradation of the misfolded protein is mediated by the proteasome.

Protein Complex Assembly and ER Exit

Protein complexes have to be fully assembled in order to be able to leave the ER.
Only correctly folded cargo is able to leave the ER.

ER Exit Sites

When proteins are properly folded, they are ready for trafficking to the Golgi apparatus and accumulate at ER exit sites.

Vesicular Transport

Transport of proteins, lipids, and other cargo between different compartments and the outside world is largely mediated by small transport carriers called transport vesicles.

Pathways

The exocytic pathway
The endocytic pathway

Steps of Vesicle-Mediated Transport

Cargo needs to be enriched in a patch of membrane.
This patch of membrane undergoes budding.
The vesicle is separated from the donor compartment by a scission reaction.
The coat disassembles.
The transport vesicle is targeted and tethered to its target compartment.
The vesicle is brought into close proximity to the target membrane (docking).
The vesicle fuses with its target compartment.

Molecular Machinery for Vesicular Transport

Cargo concentration and budding: Coat proteins and adaptor proteins
Scission: GTPases (e.g., dynamin)
Targeting and tethering: Rab proteins and tethering factors
Fusion: SNARE proteins