Protein Targeting, Secretion, and Vesicular Transport Study Notes

Introduction to Protein Targeting and Cellular Compartmentalization

• The fundamental challenge in cell biology is determining how proteins are accurately targeted to specific organelles including the endoplasmic reticulum (ER), Golgi apparatus, endosomes, lysosomes, peroxisomes, mitochondria, and chloroplasts.

• Ribosomes in the cell exist in two distinct states, though they are structurally identical:

  • Free Ribosomes: Located in the cytosol; synthesize proteins destined for the cytosol, nucleus, peroxisomes, mitochondria, and chloroplasts.

  • Membrane-Bound Ribosomes: Bound to the cytosolic surface of the ER; synthesize proteins that enter the secretory pathway.

• The Secretory Pathway covers proteins destined for:

  • The Endoplasmic Reticulum itself.

  • The Golgi Apparatus.

  • Secretory Vesicles.

  • Endosomes.

  • Lysosomes.

  • The Plasma Membrane.

  • The Extracellular Environment (secretion).

Experimental Evidence: The Pulse-Chase Experiment

• The specific pathway taken by secreted proteins was discovered using pancreatic acinar cells, which are specialized for high levels of protein secretion.

• Methodology of the Pulse-Chase Experiment:

  • Pulse: A $3\,\text{minute}$ label period using radiolabeled amino acids to tag proteins as they are synthesized.

  • Chase: Following the pulse with non-labeled amino acids to follow the "wave" of labeled proteins through the cell over time.

• Observed Timecourse and Pathway:

  • $3\,\text{minutes}$: Radiolabeled proteins are localized in the rough Endoplasmic Reticulum.

  • $7\,\text{minutes}$: Proteins have moved into the Golgi Apparatus.

  • $120\,\text{minutes}$: Proteins are found in secretory vesicles and moving toward the cell exterior.

• Definitive Pathway Conclusion: $\text{ER} \rightarrow \text{Golgi} \rightarrow \text{Secretory Vesicles} \rightarrow \text{Cell Exterior}$.

Mechanism of Co-translational Targeting to the ER

• Protein Signals: Ribosomes do not decide their destination; rather, the protein being synthesized contains specific amino acid sequences called Protein Signals.

• The ER Signal Sequence (ERSS):

  • It is typically located at the amino (N) terminus of the protein.

  • It contains a core of $\ge 12$ hydrophobic amino acids.

  • It includes a specific cleavage site for signal peptidase.

• The Signal Recognition Particle (SRP):

  • SRP is a complex consisting of $6$ polypeptides and $1$ RNA molecule ($SRP\,\text{RNA}$).

  • It features two hinges (Hinge 1 and Hinge 2) to allow for conformational changes.

  • Function: Recognizes and binds to the ERSS as it emerges from the ribosome during translation.

• Targeting Steps:

  • Step 1: The $SRP$ binds to the signal sequence on the nascent polypeptide chain.

  • Step 2: Translation is temporarily halted, and the ribosome-SRP complex binds to the $SRP$ receptor located on the ER membrane.

  • Step 3: The $SRP$ is released, and the ribosome binds to a membrane channel called the Translocon.

  • Step 4: Translation resumes, and the polypeptide is pushed through the translocon into the ER lumen (Co-translational translocation).

  • Step 5: An enzyme called Signal Peptidase cleaves the ERSS.

  • Step 6: The completed soluble protein is released into the ER lumen.

Insertion of Integral Membrane Proteins

• Integral membrane proteins possess transmembrane domains, typically characterized by an $\alpha\text{-helix}$ containing $\ge 20$ hydrophobic amino acids.

• Specific Orientation/Topologies:

  • Extracellular Domain: Facing outside the cell or inside the lumen of an organelle.

  • Transmembrane Domain: Crossing the lipid bilayer.

  • Intracellular Domain: Facing the cytoplasm.

• Mechanisms of Insertion:

  • Cleavable SS and Stop-Transfer Sequence: The N-terminus enters the lumen via a signal sequence ($\sim 12\,\text{AA}$). Translocation continues until a hydrophobic "stop-transfer" sequence ($\sim 20\,\text{AA}$) is reached, which gets anchored in the membrane.

  • Internal Non-Cleavable Signal Sequences: The signal sequence is not at the N-terminus and is not cleaved. It acts as an anchor. The orientation depends on the charges of the amino acids flanking the sequence.

  • Multiple Transmembrane Spanning Proteins: These use a series of alternating internal signal sequences and stop-transfer sequences to weave through the membrane multiple times (e.g., $G\,\text{protein-coupled receptors}$).

  • C-Terminal Transmembrane Sequences: These proteins (Tail-Anchored or TA proteins) cannot use the standard $SRP$ pathway because the signal emerges only after translation is finished. They utilize the $TRC40$ ($\text{Get3}$) pathway and the $GET1\text{-}GET2$ insertase complex in the ER membrane.

• Predictions from Primary Structure:

  • Hydrophobic Plots: Used to predict transmembrane regions. Intervals with values $\gt +2$ and lengths $\gt 19\,\text{AAs}$ suggest a transmembrane domain.

  • Case Study Comparison: $Tubulin\,\beta\,\text{chain}$ (cytosolic) vs. $Aquaporin\text{-}1$ (plasma membrane). Aquaporin shows multiple distinct hydrophobic peaks on a plot, while tubulin does not.

Protein Processing and Quality Control in the ER

• Proteolytic Processing: Example: Preproinsulin (with signal sequence) is converted to Proinsulin (cleavage of SS). Proinsulin then undergoes further cleavage of the "connecting polypeptide" to form mature active Insulin.

• Formation of Disulfide Bonds: Facilitated by $PDI$ (Protein Disulfide Isomerase). The ER lumen is an oxidizing environment allowing $\text{-S-S-}$ bonds, whereas the cytosol is reducing ($\text{-SH}$ state).

• Protein Folding: Assisted by ER-resident chaperones like $BiP$ (an $Hsp70$ family member). Chaperones prevent protein aggregation.

• N-linked Glycosylation:

  • A preformed oligosaccharide is transferred from a dolichol lipid carrier to an Asparagine (N) residue on the peptide.

  • Three glucose residues are initially present and are subsequently removed by enzymes.

• Quality Control and the Calnexin/Calreticulin Cycle:

  • Calnexin and Calreticulin are chaperones that bind to glycoproteins containing a single glucose.

  • If the protein is misfolded, $UDP\text{-Glucosyltransferase}$ adds a glucose back to force the protein through another cycle of folding.

  • Persistent misfolding leads to the removal of mannose residues and retro-translocation of the protein to the cytosol for degradation by the proteasome (Ubiquitin-dependent mechanism).

• The Unfolded Protein Response (UPR):

  • Triggered by the accumulation of misfolded proteins.

  • Effects: General inhibition of protein synthesis, increased expression of chaperones ($BiP$, $PDI$, $Calnexin$), and increased proteasomal activity.

• Addition of GPI Anchors:

  • Proteins are attached to Glycosylphosphatidylinositol (GPI) anchors at their C-terminus.

  • Critical Topology: GPI-anchored proteins always face the extracellular environment (or organelle lumen), never the cytosol.

The Golgi Apparatus and Vesicular Traffic

• Organization: Composed of flattened sacs called cisternae, organized into:

  • $cis\,\text{Golgi}$ network (receiving side from ER).

  • Golgi stack (medial and trans cisternae).

  • $trans\,\text{Golgi}$ network (TGN; sorting and shipping side).

• Models of Golgi Transport:

  • Stable Cisternae Model: Vesicles move proteins between stationary cisternae.

  • Cisternal Maturation Model: The cisternae themselves migrate and mature from cis to trans.

• Sorting and Retrieval Signals:

  • ER Export Signals: Di-hydrophobic or Di-acidic sequences for integral proteins.

  • ER Retrieval (Retention) Signals: Used for resident ER proteins that escape to the Golgi.

  • $KDEL$: Sequence for soluble ER proteins at the C-terminus.

  • $KKXX$: Sequence for membrane ER proteins.

• Golgi Glycosylation Processing: Sequential modification of N-linked oligosaccharides occurs as proteins move through the stack (e.g., removal of mannose, addition of $N\text{-acetylglucosamine}$, fucose, galactose, and sialic acid).

• Targeting to Lysosomes:

  • Proteins destined for lysosomes are recognized by a "Signal Patch."

  • They are modified by the phosphorylation of mannose residues to create $Mannose\text{-}6\text{-phosphate}$.

Mechanisms of Vesicular Transport

• Budding and Coat Proteins:

  • Budding is driven by the assembly of coat proteins on the cytosolic side.

  • $COPII$: Mediate anterograde traffic (ER to Golgi).

  • $COPI$: Mediate retrograde traffic (Golgi to ER).

  • Clathrin: Mediate traffic between the TGN, endosomes, and the plasma membrane.

  • Adaptin: Mediates the interaction between cargo receptors (integral membrane proteins) and the coat proteins.

• Fusion and SNAREs:

  • Targeting specificity is ensured by SNARE proteins.

  • $v\text{-SNAREs}$: Located on the vesicle membrane.

  • $t\text{-SNAREs}$: Located on the target membrane.

• Transport Mechanisms: Movement of vesicles is facilitated by motor proteins along microtubules.

  • Kinesin: Generally moves toward the plus ($+$) end (plasma membrane).

  • Dynein: Generally moves toward the minus ($-$) end (nucleus/ER).