Cell Biology Protein Transport Quiz

MPharm Programme: Protein Sorting and Secretion

• Course: MPharm
• Module: PHA114
• Topic: Protein Sorting and Secretion
• Week: 26

Generation of Proteins

• Proteins are generated in the nucleus via transcription.
• mRNA is transported to the cytosol for translation.
• Translation occurs on ribosomes.

Endocytosis (IN) and Exocytosis (OUT)

• Endocytosis brings molecules into the cell.
• Exocytosis releases molecules from the cell.
• Processes involve the endoplasmic reticulum, lysosomes, golgi, and transport vesicles.

Where Do Proteins Do Their Job?

• Proteins are used throughout the cell (nucleus, cytosol organelles, cell membrane).
• A precise, regulated system moves proteins to their site of action.

Overview of Protein Sorting

• mRNA is transcribed in the nucleus and translated in the cytosol to create proteins.
• Non-secretory proteins lack an ER signal; they are translated on free ribosomes.
• Secretory proteins (including those destined for membranes) are translated on ribosomes that are bound to the ER.
• Proteins then travel through the ER, the Golgi complex, and then to their final destination, either outside the cell or in specific membrane-bound organelles,.

Protein Secretion (Co-translational Translocation)

• Signal sequence on mRNA is translated.
• Signal sequence is bound by signal recognition particle (SRP).
• SRP targets ribosome to SRP receptor on ER membrane (requires GTP).
• Translocon opens.
• Signal sequence and growing peptide chain insert.
• GTP hydrolysis; signal sequence release.
• Polypeptide chain passes through translocon.
• Signal sequence cleavage by membrane-bound peptidase.
• Polypeptide elongation and folding.
• Protein release into the ER lumen.
• Ribosome release.

Protein Secretion

• All eukaryotic cells use the same secretory pathway for protein synthesis and sorting.
• Proteins are sorted to their destination in the endoplasmic reticulum membrane or lumen.
• Proteins are sorted or translocated across the ER membrane and then transferred to the golgi via transport vesicles.
• Proteins are transported through the golgi complex.
• Proteins travel to their final destination (plasma membrane, lysosomes, or cell surface) via vesicles.

Protein Structure

• Proteins destined for specific locations in the cell have an N-terminal signal sequence.
• This directs ribosomes to the endoplasmic reticulum.
• The signal sequence directs the protein to the ER via the SRP.

Common Signal Sequences

• Specific sequences (e.g., KDEL, KKXX) direct proteins to specific intracellular compartments.

The Cell Membrane

• The cell membrane is a complex structure with lipids (incl. phospholipids, cholesterol), proteins and carbohydrates.

Inserting Proteins into Membranes

• Ribosomes attach to the translocon on the ER membrane.
• Protein translation continues through the translocon.
• Hydrophobic stretches are identified and are incorporated into the membrane.
• The protein is released into the membrane.
• This process establishes the protein's orientation in the membrane, with one side facing the cytosol and the other side facing the interior of the ER or extracellular space.

Insertion of proteins into membranes

• Protein orientation in the membrane is permanent.
• Proteins are translocated or transferred to the ER until a hydrophobic membrane-crossing domain is seen.
• This serves as a signal and the protein is inserted in the ER membrane.

The secretory pathway

• The pathway is used to send soluble proteins and cell-membrane-bound proteins.
• Final destinations might be outside the cell or into the plasma membrane, lysosomes, or other organelles.

Secretory Proteins

• Newly generated proteins are translocated into the ER lumen.
• Vesicles fuse with the cis-Golgi.
• Proteins may be sent back to the ER.
• Transport from the golgi to the ER is via retrograde vesicles.
• Non-vesicular transport may also occur.

Secretory Proteins

• Proteins that are to be secreted and those destined for the plasma membrane or lysosomes are sent to the trans-Golgi network. 
• Proteins are sent to the cell membrane via secretory vesicles via regulated secretion.

Vesicular Trafficking

• Vesicles are used to move cargo between cellular compartments;
• Proteins remain oriented such that their orientation in the the membrane is maintained.

Types of Vesicle

• COP II vesicles transport proteins from the rough ER to the Golgi.
• COP I vesicles transport in retrograde direction between the Golgi and the ER.
• Clathrin-coated vesicles transport proteins from the plasma membrane to endosomes.

Molecular Mechanisms of Vesicle Transport

• Vesicle budding is initiated by the recruitment of small GTP proteins which results in an invagination.
• Membrane proteins and cargo proteins are then recruited into the budding vesicle.
• The vesicle membranes then fuse with target membranes.

Molecular Mechanisms of Vesicle transport (vesicle docking)

• Vesicle docks to a target membrane through the interaction of SNARE proteins.
• v-SNARE and t-SNARE pairs.
• SNARE proteins promote fusion and membrane fusion with the correct membrane.
• NSF protein dismembers SNARE complex;
• ATP needed.

The KDEL receptor :Retrieval of ER resident proteins

• ER proteins with a C-terminal KDEL signal sequence are retrieved from the Golgi to the ER.
• COP I vesicles transport the proteins back to the ER.

The Endocytic Pathway

• Pathway used to bring proteins and other molecules into the cell.
• Examples include uptake, and removal of cell-surface proteins.

Clatherin-coated vesicle formation in endocytosis

• Vesicles form at the plasma membrane to bring materials into cells and are coated with clathrin proteins.

Internalisation of low density lipoprotein (LDL)

• LDL particles are taken up by cells via receptor-mediated endocytosis.
• LDL receptors bind to LDL particles, causing vesicle formation and internalization.
• LDL is then transported to lysosomes where it is degraded.

Protein Modifications

• Membrane and soluble secretory proteins are glycosylated and stabilized by Cys-Cys bonds, they are assembled into multi-subunit conformations and cleaved to their active conformation.

Glycosylation of Proteins

• Carbohydrate chains are added to proteins via O-linked or N-linked bonds.
• O-linked glycosylation adds sugars to the hydroxyl groups of serine or threonine.
• N-linked glycosylation adds sugars to the amide nitrogen of asparagine.

Generation of N-linked core glycans

• Dolichol phosphate (DP) is a hydrophobic lipid.
• First sugar (N-acetylglucosamine) is added to DP.
• Remaining sugars added by glycosidic-linkages.
• Enzymes are found in the ER membrane and will flip the DP with the residue across the membrane.
• The 14-residue precursor is transferred and trimmed.

Disulfide bond formation & re-arrangement

• Disulfide bonds are formed and re-arranged in the ER lumen.
• Cys-Cys bonds help stabilise protein structure in secretory proteins and those in the membrane.
• PDI is involved, with its oxidized and reduced forms.
• Ero1 regenerates PDI.

Chaperones help fold proteins

• Chaperones assist proteins folding correctly inside the ER lumen.
• They prevent aggregation.
• They typically require ATP.

Unfolded Protein Response (UPR)

• Response activated when there are misfolded proteins in the ER lumen
• Pathway and the activity of Ire1 protein.
• Unfolded proteins cause BiP to release from Ire1, which activates it.
• Cleavage of Hac1 mRNA occurs, which allows Hac1 translation and the expression of more chaperones (and other proteins).

Studying protein sorting and secretion

• Microscopy and pulse-chase experiments are two methods used for studying protein sorting and secretion.
• Genetic analysis of secretory pathway mutants is one way to analyze different stages of the pathway.
• Studying these mutants provides insight into the individual roles of proteins during the secretory pathway.

Summary

• Protein folding, modification, and packaging is a complex and highly regulated process.
• Membrane-bound vesicles are used to transport proteins.

Reference

• Lodish H et al., Molecular Cell Biology.