Lecture 5 Notes: Protein Targeting and the Endoplasmic Reticulum
Parkinson's Disease (PD)
Genetic Factors:
Autosomal recessive: Parkin
Loss-of-function mutations in the Parkin gene.
Encodes an E3 ubiquitin ligase involved in targeting proteins for degradation via the ubiquitin-proteasome system.
Mutations lead to the accumulation of misfolded or damaged proteins, contributing to neuronal dysfunction.
Autosomal dominant: LRRK2, α-Synuclein
LRRK2 (Leucine-rich repeat kinase 2):
Gain-of-function mutations in the LRRK2 gene
Encodes a kinase involved in various cellular processes, including vesicle trafficking and autophagy.
Mutations enhance kinase activity leading to impaired cellular function.
α-Synuclein:
Missense mutations, multiplications, and point mutations in the α-Synuclein gene.
Encodes a protein abundant in neurons, especially at presynaptic terminals.
Mutations promote aggregation and formation of Lewy bodies, a pathological hallmark of PD.
Genetic risk factors: α-Synuclein, LRRK2, Tau
Specific variants in these genes increase susceptibility to developing PD, even in the absence of clear Mendelian inheritance patterns.
Environmental Factors:
Toxins (e.g., rotenone, paraquat)
Exposure to environmental toxins like rotenone (pesticide) and paraquat (herbicide) has been linked to an increased risk of developing PD.
These toxins interfere with mitochondrial function, leading to oxidative stress and neuronal damage.
Aging
Aging is the biggest risk factor for PD.
Cellular and molecular changes such as accumulation of oxidative damage, impaired protein degradation, and mitochondrial dysfunction, increase with age, contributing to the pathogenesis of PD.
Sporadic PD:
Complex I activity
Dysfunction of mitochondrial complex I.
Leads to reduced ATP production and increased production of reactive oxygen species (ROS).
Oxidative stress
Imbalance between the production of ROS and the ability of the cell to detoxify these harmful molecules.
ROS can damage cellular components, including lipids, proteins, and DNA, leading to neuronal dysfunction and death.
Bioenergetics
Impairments in cellular energy metabolism affect neuronal function and survival.
Quality control
Dysfunction in protein quality control mechanisms, such as the ubiquitin-proteasome system (UPS) and autophagy.
Leads to the accumulation of misfolded and aggregated proteins, contributing to neuronal toxicity.
homeostasis
Disruption of calcium homeostasis can lead to excitotoxicity and neuronal damage.
Biogenesis
Abnormalities in the formation and maintenance of cellular structures and organelles.
Dynamics and transport (CHNI, AMIN)
Disturbances in cellular transport processes, including the movement of molecules and organelles within neurons.
Protein Transport Overview
Proteins are transported to various organelles including:
Nucleus
Plastids
Mitochondria
Peroxisomes
Endoplasmic Reticulum (ER)
Golgi
Lysosome
Early and Late Endosomes
Secretory Vesicles
Cell Exterior
Peroxisomes
Peroxisome Import:
A three-amino acid sequence at the C-terminal site is required for import.
Proteins destined for the peroxisome matrix typically contain a peroxisomal targeting signal type 1 (PTS1) at their C-terminus, consisting of the tripeptide Ser-Lys-Leu (SKL) or a conservative variant.
Even oligomeric proteins are imported from the cytosol without unfolding.
Unlike transport into other organelles such as mitochondria, peroxisomes can import folded proteins and even large oligomeric complexes.
Mechanism:
Binding of cargo to PEX5 in the cytosol.
The PTS1 receptor, PEX5, binds to the cargo protein in the cytosol.
Docking at the peroxisome membrane involving proteins like PEX14, PEX13, PEX12, PEX10, and PEX22.
The PEX5-cargo complex docks at the peroxisome membrane via interactions with peroxins such as PEX14 (the main docking receptor), PEX13, PEX12, PEX10, and PEX22.
Translocation across the membrane.
The mechanism of translocation across the peroxisome membrane is not fully understood, but it involves the formation of a transient pore or channel that allows the passage of the cargo protein.
Receptor recycling involving PEX4 and PEX8.
After releasing the cargo into the peroxisome matrix, PEX5 is recycled back to the cytosol in a process that requires PEX4 (an E3 ubiquitin ligase) and PEX8.
Localized Translation:
Peroxisomes utilize localized translation to ensure correct insertion of hydrophobic proteins into their membranes.
mRNAs encoding peroxisomal membrane proteins (PMPs) are selectively targeted to the vicinity of peroxisomes, where they are translated.
Peroxisomal membrane mRNAs are found in proximity to peroxisome membranes.
BioID Proximity-Dependent Biotinylation:
A method used to detect mRNA found in proximity to peroxisomes.
Biotin ligase is attached to a peroxisome membrane protein.
AviTag is fused to a large ribosomal subunit.
Biotin is added, leading to biotinylation of nearby ribosomes and transcripts.
Streptavidin pulldown is used to isolate biotinylated proteins.
Sequencing of protected transcripts.
Endoplasmic Reticulum (ER)
General Information:
All eukaryotic cells have an ER.
The endoplasmic reticulum (ER) is a continuous network of membranes that extends throughout the cytoplasm of eukaryotic cells.
It constitutes about half of the cell membrane.
The ER membrane is highly extensive, accounting for approximately 50% of the total membrane surface area in a typical eukaryotic cell.
The ER labyrinth is continuous with the nuclear membrane.
The outer nuclear membrane is continuous with the ER membrane, providing a direct connection between the nucleus and the ER.
It has a central role in protein and lipid biosynthesis and serves as an intracellular storage.
The ER is the primary site of synthesis for secreted and membrane proteins and is also involved in the synthesis of lipids and steroids. Furthermore, the ER functions as a major intracellular calcium storage compartment, regulating calcium homeostasis.
Structural and Functional Diversity:
Rough ER: Contains ribosomes and is involved in protein synthesis.
The rough ER (RER) is studded with ribosomes on its cytoplasmic surface, giving it a "rough" appearance. These ribosomes are actively engaged in protein synthesis, particularly of proteins destined for secretion or insertion into membranes.
Smooth ER: Lacks ribosomes and is involved in lipid synthesis and calcium storage.
The smooth ER (SER) lacks ribosomes and is primarily involved in lipid synthesis, including the production of phospholipids, cholesterol, and steroid hormones. It also plays a crucial role in calcium storage and detoxification.
Cisternae: The flattened, membrane-enclosed sacs or tube-like structures that make up the ER.
The ER membrane is organized into a network of interconnected tubules and flattened sacs called cisternae, which provide a large surface area for biochemical reactions.
Transitional ER: Regions where transport vesicles bud off to carry proteins and lipids to the Golgi apparatus.
Transitional ER (tER) sites are specialized regions of the ER where transport vesicles bud off to shuttle proteins and lipids to the Golgi apparatus for further processing and sorting.
Co-translational vs. Post-translational Translocation:
Co-translational translocation: Occurs while the protein is being synthesized by the ribosome.
In co-translational translocation, the protein is translocated into the ER lumen as it is being synthesized by the ribosome. This process is mediated by the signal recognition particle (SRP) and the translocon channel.
Post-translational translocation: Occurs after the protein has been fully synthesized.
In post-translational translocation, the protein is synthesized in the cytoplasm and then translocated into the ER lumen after its synthesis is complete. This process requires the assistance of chaperone proteins such as BiP.
Fractionation of Organelles:
Sucrose gradient centrifugation can be used to separate rough and smooth ER microsomes.
Microsomes are vesicles derived from the ER that can be separated based on their density using sucrose gradient centrifugation. Rough microsomes (derived from RER) are denser than smooth microsomes (derived from SER) due to the presence of ribosomes.
Protein Synthesis in the ER:
The ER synthesizes both transmembrane and water-soluble proteins.
The ER is responsible for the synthesis of a wide variety of proteins, including transmembrane proteins that reside in the ER membrane and water-soluble proteins that are secreted into the ER lumen.
Proteins destined for the lysosomes, secretion, cell membrane and the Golgi apparatus are synthesized within the ER.
Signal Hypothesis (Gunter Blobel):
Secretory proteins are synthesized with an amino-terminal extension (signal peptide).
The signal hypothesis proposes that secretory proteins contain a signal peptide, a short amino acid sequence at their N-terminus that directs them to the ER membrane.
The signal peptide is recognized by a cytosolic factor and targets the protein to the ER.
The signal peptide is recognized by the signal recognition particle (SRP), a cytosolic ribonucleoprotein that binds to the signal peptide and escorts the ribosome-mRNA complex to the ER membrane.
Experimental Support for the Signal Hypothesis:
Precursor-product relationship: The signal peptide is cleaved off during translocation into the ER, resulting in a mature peptide.
Experimental evidence for the signal hypothesis comes from studies showing that secretory proteins are initially synthesized as larger precursor proteins with a signal peptide, which is subsequently cleaved off by a signal peptidase in the ER lumen to produce the mature protein.
Why Co-translational Translocation?
Precursor proteins tend to aggregate in the cytosol prior to translocation.
Two strategies to prevent aggregation:
Coupling translocation with protein synthesis (co-translational translocation).
Recruiting cytosolic chaperones to prevent aggregation (post-translational translocation).
SRP (Signal Recognition Particle):
A hydrophobic motif (signal sequence) is recognized by the SRP that mediates ER translocation.
The signal recognition particle (SRP) recognizes and binds to the signal sequence, a hydrophobic motif present at the N-terminus of proteins destined for the ER.
The SRP brings the ribosome to the ER membrane.
The SRP escorts the ribosome-mRNA complex to the ER membrane by binding to the SRP receptor on the ER surface.
SRP Mechanism:
Binding of SRP to the signal peptide causes a pause in translation.
Binding of SRP to the signal peptide induces a pause in translation, preventing the premature folding of the protein in the cytosol.
The SRP receptor in the rough ER membrane targets the ribosome to the translocon.
The SRP receptor, located on the ER membrane, targets the ribosome-SRP complex to the translocon, a protein channel in the ER membrane.
Translation continues, and translocation begins.
Once the ribosome is docked at the translocon, translation resumes, and the polypeptide chain is threaded through the translocon into the ER lumen.
Translocator/Translocon:
A protein channel in the ER membrane through which polypeptide chains are translocated.
The translocon is a protein-conducting channel in the ER membrane composed of the Sec61 complex.
Opens from the top and sideways to form a pore and allow hydrophobic portions of proteins to partition into the lipid bilayer.
The translocon can open laterally to allow hydrophobic transmembrane domains of proteins to exit the channel and partition into the lipid bilayer.
Signal Sequence Recognition:
The signal sequence is recognized twice during translocation.
The signal sequence is initially recognized by the SRP in the cytosol and subsequently by the translocon in the ER membrane.
Types of Membrane Proteins:
Type I: N-terminus in the ER lumen, C-terminus in the cytosol, has a cleaved signal sequence.
Type I membrane proteins have a cleaved signal sequence and a stop-transfer anchor sequence, resulting in the N-terminus being located in the ER lumen and the C-terminus in the cytoplasm.
Type II: N-terminus in the cytosol, C-terminus in the ER lumen, has an internal signal sequence.
Type II membrane proteins have an internal signal sequence that also functions as a membrane anchor, resulting in the N-terminus being located in the cytoplasm and the C-terminus in the ER lumen.
Type III: N-terminus in the ER lumen, C-terminus in the cytosol, has an internal signal sequence.
Type III membrane proteins have an internal signal sequence that also functions as a membrane anchor, resulting in the N-terminus being located in the ER lumen and the C-terminus in the cytoplasm.
Type IV: Multi-pass transmembrane proteins.
Type IV membrane proteins have multiple transmembrane domains and can have varying orientations of their N- and C-termini.
GPI-linked protein: Attached to the membrane via a glycosylphosphatidylinositol (GPI) anchor.
GPI-linked proteins are attached to the ER membrane via a glycosylphosphatidylinositol (GPI) anchor, a lipid modification added to the C-terminus of some proteins.
Translocation of Single-Pass Transmembrane Proteins:
N-terminal signal sequence (Type I): A stop-transfer sequence interacts with the translocon, opening the seam and discharging the protein laterally into the lipid bilayer.
Type I single-pass transmembrane proteins utilize an N-terminal signal sequence to initiate translocation, followed by a stop-transfer anchor sequence that halts translocation and anchors the protein in the membrane.
Internal signal sequence (Types II and III): The orientation of the protein depends on the distribution of positively charged amino acids around the signal sequence.
The orientation of Type II and Type III single-pass transmembrane proteins is determined by the distribution of positively charged amino acids flanking the internal signal sequence, with the side having more positive charges oriented towards the cytoplasm.