Protein Sorting and Vesicular Transport Flashcards
Endoplasmic Reticulum (ER) Protein Targeting and Synthesis
ER Co-translational Translocation
Proteins destined for the ER are generally transported across the membrane while they are still being synthesized (co-translational translocation).
These proteins possess an N-terminal signal sequence which facilitates the docking of the synthesizing ribosome onto the ER.
The entry of the protein across the ER membrane occurs simultaneously as the polypeptide chain is synthesized.
RNA and Protein Expression in the Rough ER
The rough ER consists of ER membranes and polyribosomes.
RNAs isolated from the rough ER encode for:
Soluble secreted proteins.
ER membrane proteins.
Plasma membrane proteins.
Proteins destined for the lysosome or Golgi apparatus.
Cytosolic Protein Synthesis
Ribosomal proteins are translated in the cytosol by cytosolic ribosomes, as they are cytoplasmic components.
Experimental Manipulation of ER Signal Sequences
Addition to Cytosolic Protein: Adding an ER signal sequence to the N-terminal of a normally cytosolic protein will cause it to be transported into the ER lumen.
Charge Alteration: Changing hydrophobic amino acids in an ER signal sequence to charged amino acids prevents recognition, causing the protein to remain in the cytosol.
Sequence Variation: Replacing hydrophobic amino acids with different hydrophobic amino acids still allows for ER delivery. The physical distribution of hydrophobicity is the critical factor, not the specific sequence of amino acids.
C-terminal Relocation: Moving the N-terminal ER signal sequence to the C-terminal end prevents ER entry. Since the C-terminus is synthesized last, the Signal-Recognition Particle (SRP) cannot recognize the sequence in time to carry the ribosome to the ER.
Soluble ER Proteins (Example: Fuzzy)
Soluble proteins in the ER lumen (like Fuzzy) typically have N-terminal signal sequences (not C-terminal).
Multiple ribosomes can bind to a single mRNA molecule during translation (polyribosomes).
Hydrophobic stop-transfer sequences are present in membrane-inserted proteins but are absent in soluble proteins.
Once cleaved, the signal peptide is ejected into the ER membrane and subsequently degraded.
Protein Topology and Translocation Mechanisms
ER Lumen vs. Plasma Membrane Proteins
Proteins residing in the ER lumen are synthesized by membrane-bound ribosomes.
These proteins may eventually be secreted into the extracellular space or reside in other organelles of the endomembrane system.
Plasma membrane proteins originate from proteins embedded in the ER membrane, not from the ER lumen.
Mechanism of Targeting
ER-destined proteins are translated by cytosolic ribosomes and targeted to the ER as soon as the signal sequence emerges during the translation process.
Components of ER Transport and Their Locations
Signal-Recognition Particle (SRP): Cytosol.
SRP Receptor: ER Membrane.
Active site of Signal Peptidase: ER Lumen.
Protein Translocator: ER Membrane.
mRNA: Cytosol.
Internal Stop-Transfer Sequences
For a protein with an N-terminal signal sequence and an internal stop-transfer sequence, the N-terminus initiates translocation through the channel.
When the stop-transfer sequence enters the translocator, the channel discharges both the signal sequence and the stop-transfer sequence sideways into the lipid bilayer.
The signal sequence is cleaved, leaving the protein anchored by the hydrophobic stop-transfer sequence as a single-pass transmembrane protein.
Competition of Signal Sequences
Nuclear Import vs. ER Import: The protein enters the ER because ER targeting is co-translational, whereas nuclear import occurs post-translationally with fully folded proteins.
Nuclear Export vs. Mitochondrial Import: The protein enters the mitochondria; for export to work, the protein would have had to enter the nucleus first via an import signal.
Mitochondrial Import vs. ER Retention: The protein enters the mitochondria. ER retention requires the protein to enter the ER first; without an ER import signal, the retention signal is never activated.
Multi-pass Transmembrane Proteins
Deleting an N-terminal signal sequence or mutating its hydrophobic residues to charged residues can cause the next segment to act as an internal start-transfer signal, inverting the protein's orientation.
Mutated signal sequences on the cytosolic side are not cleaved because signal peptidase is located only within the ER lumen.
Vesicular Transport and the Secretory Pathway
Protein Traversal Pathway
Proteins destined for the plasma membrane follow the order: ER Golgi Plasma Membrane.
Transport Terminology and Systems
Proteins exit the cell via the secretory or exocytic pathways.
Fluids and macromolecules enter via the endocytic pathway.
Transport vesicles link organelles within the endomembrane system.
Vesicle Budding Components (Golgi)
Clathrin: Acts at the cytosolic surface to help shape the vesicle; released after budding.
Adaptins: Select cargo by capturing cargo receptors that bind specific molecules; they also interact with clathrin.
Dynamin: Required for the budding process (pinching off). Absence of dynamin results in the formation of coated pits that cannot mature into vesicles.
Directing and Fusing Transport Vesicles
Rabs: GTP-binding proteins involved in the recognition and docking of vesicles to target membranes.
Tethering Proteins: Initial interaction factors for docking.
SNAREs (v-SNARES and t-SNARES): Mediate recognition and catalyze membrane fusion by pulling the vesicle and target membranes together.
Adaptins in Targeting: Not involved in targeting; they are removed during uncoating.
The Fusion Process
Vesicle docking does not always trigger immediate fusion.
Fusion requires the displacement of water molecules from the hydrophilic membrane surfaces.
GTP hydrolysis by Rab proteins is essential for docking/tethering, but does not provide the direct energy for fusion.
SNARE-Mediated Fusion Specifics (Yeast Vacuole Study)
Maximal fusion occurs when vesicles carry both v-SNAREs and t-SNAREs.
Fusion is possible as long as complementary SNAREs are present between the two vesicles even if one lacks a specific type.
Fusion is minimal or fails if both vesicles lack v-SNAREs or both lack t-SNAREs.
Protein Modification and ER Quality Control
Glycosylation (N-linked)
Oligosaccharides are attached to secreted glycoproteins at the nitrogen atom of the asparagine side chain within the sequence sequence Asn-X-Ser/Thr.
A branched -sugar oligosaccharide is added as a single unit, then modified by enzymes in the ER and Golgi before the protein reaches the plasma membrane.
Disulfide Bond Formation
Occurs in the ER lumen as it is an oxidizing environment.
Does not occur in the cytosol because it is a reducing environment.
Formed by the oxidation of cysteine side chain pairs, catalyzed by ER lumen enzymes.
Stabilizes protein structure.
ER Retention and Secretion
Proteins with an ER retention signal are captured in the Golgi and returned to the ER.
Removal of this signal causes the protein to follow the default secretory pathway toward the extracellular space.
ER Quality Control System
Chaperone proteins facilitate proper folding and retain misfolded proteins or unassembled complexes within the ER.
Misfolded proteins are exported from the ER into the cytosol for degradation (at the proteasome); they are not degraded within the ER lumen.
Unfolded Protein Response (UPR)
Triggered when sensors in the ER (not the cytoplasm) detect misfolded proteins.
Leads to increased production of ER membrane and chaperone proteins.
Involves the cytoplasmic activation of gene regulatory proteins to expand the ER's capacity.