Lecture 6 Summary: Protein Translocation, Glycosylation, and Quality Control in the ER

Types of Membrane Proteins

• Membrane proteins are categorized based on their orientation within the lipid bilayer and their interactions with it. These proteins serve diverse functions, from signaling and transport to maintaining cellular structure.

Type I Membrane Proteins

• Characteristics: These are single-pass transmembrane proteins characterized by having their N-terminus located in the exoplasmic space while their C-terminus is in the cytosol. The orientation is crucial for their function, often involving receptor or enzymatic activities.

• Examples:

  • LDL receptor: Involved in cholesterol uptake.

  • Influenza HA protein: Mediates viral entry into host cells.

  • Insulin receptor: Receptor tyrosine kinase that binds insulin.

  • Growth hormone receptor: Activates signaling pathways for growth and metabolism.

Type II Membrane Proteins

• Characteristics: Single-pass transmembrane proteins that have the N-terminus in the cytosol and the C-terminus in the exoplasmic space. This orientation is vital for their roles in various cellular processes.

• Examples:

  • Asialoglycoprotein receptor: Mediates endocytosis of glycoproteins.

  • Transferrin receptor: Involved in iron uptake.

Type III Membrane Proteins

• Characteristics: Single-pass transmembrane proteins with a specific orientation that is critical for their enzymatic function within cellular compartments, such as the Golgi apparatus.

• Examples:

  • Cytochrome P450: Enzymes involved in drug metabolism and synthesis of various molecules.

  • Golgi galactosyltransferase: Catalyzes the transfer of galactose to proteins in the Golgi.

  • Golgi sialyltransferase: Catalyzes the transfer of sialic acid to proteins in the Golgi.

Type IV Membrane Proteins

• Characteristics: Multi-pass transmembrane proteins that cross the membrane multiple times. This complex topology is essential for forming channels or transporters.

• Examples:

  • G protein-coupled receptors: Mediate cellular responses to hormones and neurotransmitters.

  • Glucose transporters: Facilitate glucose uptake into cells.

  • Voltage-gated Ca^{2+} channels: Regulate calcium ion flow across the cell membrane.

  • ABC small molecule pumps: Transport various molecules across membranes.

  • CFTR (Cl^{-}) channel: Regulates chloride ion transport in epithelial cells.

  • Sec61: Protein-conducting channel in the ER membrane.

GPI-Linked Proteins

• Characteristics: Proteins anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor. These proteins are typically found on the cell surface and are involved in signaling and adhesion.

• Examples:

  • Plasminogen activator receptor: Involved in cell surface signaling.

  • Fasciclin II: Cell adhesion molecule in neuronal development.

Translocation of Double Pass Transmembrane Protein

• Double-pass transmembrane proteins contain an internal signal sequence, which functions as a start-transfer sequence, initiating their insertion into the ER membrane.

• A stop-transfer sequence then halts the transfer process, ensuring the protein is embedded in the ER membrane with two transmembrane domains.

• Hydrophobic start-transfer and stop-transfer peptides are crucial for the correct orientation of the protein within the lipid bilayer.

• The translocator protein (Sec61 complex) facilitates the insertion of these transmembrane segments into the ER membrane.

Translocation of Multipass Membrane Protein

• Multipass membrane proteins have multiple start and stop transfer sequences, enabling them to weave in and out of the ER membrane multiple times.

• The alternating start and stop signals lead to the protein spanning the membrane multiple times, forming complex three-dimensional structures.

Post-Translational Translocation of Tail-Anchored Proteins

• Pre-targeting Complex: Tail-anchored proteins are targeted to the ER membrane after translation, as their hydrophobic C-terminal tail is not recognized by SRP.

• Recognition: The Get3 ATPase recognizes the tail-anchored protein, shielding its hydrophobic tail from the aqueous environment.

• Targeting: The Get3 complex interacts with Get1 and Get2 in the ER membrane, facilitating insertion of the tail-anchored protein.

• Release: The tail-anchored protein is released into the ER membrane, where it can perform its function.

• Recycling: The Get3 ATPase is recycled to bind more tail-anchored proteins.

SRP Independent ER Translocation

• Some proteins translocate to the ER independently of the Signal Recognition Particle (SRP), often relying on alternative mechanisms.

• This includes proteins with mildly hydrophobic signal sequences, structurally inadequate short secretory signals, and tail-anchored proteins.

• Chaperone proteins such as Hsp40s, Hsp70s, and Calmodulin are involved in targeting and chaperoning these proteins to the ER membrane.

• The Sec62-Sec63 complex and Kar2/BiP assist in translocation by facilitating the opening of the Sec61 translocon.

Post Translational Translocation

• ATP-driven cycles of binding and releasing of BiP (Binding Immunoglobulin Protein) drive the pulling of post-translated proteins into the ER lumen, ensuring proper folding and preventing aggregation.

Protein Processing in the ER

• After translocation to the ER, proteins undergo folding, post-translational modifications such as glycosylation, and quality control to ensure they are correctly processed.

N-Glycosylation in the ER

• N-Glycosylation begins in the ER with the addition of a core oligosaccharide to asparagine residues (Asn) in the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline.

• The oligosaccharide (Glc3Man9GlcNAc2) is initially assembled on dolichol diphosphate, a lipid molecule embedded in the ER membrane.

• The flippase then flips the dolichol-linked oligosaccharide to the ER lumen side, where it can be transferred to the protein.

N-Glycosylation Mediates Protein Folding

• N-Glycosylation plays a crucial role in protein folding by associating with chaperone proteins like calreticulin and calnexin.

• Glucosyltransferase adds a glucose residue to partially folded proteins, and the removal of glucose residues by glucosidases is a critical part of the quality control process.

• ER-associated degradation (ERAD) is initiated if the protein misfolds and cannot be properly corrected by chaperone proteins.

N-Glycosylation in the Golgi

• N-Glycosylation continues in the Golgi, where further modifications occur, leading to a diverse array of glycan structures.

• Various enzymes in the Golgi add or remove sugars such as N-acetylglucosamine, mannose, glucose, galactose, and sialic acid, tailoring the glycosylation pattern to the protein's specific function and destination.

Glycoprotein Processing

• A unique composition of enzymes is present along the early secretory pathway to sequentially process glycoproteins, ensuring proper folding, stability, and trafficking.

Biochemical Deglycosylation Assay

• Biochemical deglycosylation assays, using enzymes like PNGaseF and Endoglycosidase H (EndoH), are valuable tools to determine protein glycosylation patterns and study glycoprotein processing.

• PNGaseF removes almost all N-linked oligosaccharides, providing information about the total glycosylation status of the protein.

• EndoH is specific for high-mannose, ER-type N-glycans, allowing researchers to distinguish between ER-resident and Golgi-modified glycoproteins.

Protein Glycosylation to Determine Protein Localization

• Protein glycosylation patterns can be used to determine protein localization within the cell, as different compartments have distinct glycosylation enzymes.

ER Stress

• ER stress is sensed by transmembrane sensors (IRE1, ATF6, PERK) in the ER membrane, which detect the accumulation of unfolded or misfolded proteins.

• Activation of these sensors leads to the initiation of the unfolded protein response (UPR), a cellular mechanism designed to restore normal function by enhancing the protein-folding capacity and promoting degradation of misfolded proteins.