Protein Targeting, the Secretory Pathway, and Vesicular Transport

Intracellular Protein Targeting and Organelles

• Proteins must be localized to specific compartments to function correctly within the eukaryotic cell.

• Key organelles involved in protein targeting include:

  • Endosome: Involved in sorting and routing material internalized by the cell.

  • Cytosol: The fluid portion of the cytoplasm where many proteins remain after synthesis.

  • Peroxisome: Specialized for oxidative reactions.

  • Lysosome: The primary digestive organelle containing acid hydrolases.

  • Endoplasmic Reticulum (ER): The entry point for the secretory pathway.

  • Golgi Apparatus: Involved in modifying, sorting, and packaging proteins.

  • Mitochondria and Chloroplasts: Organelles with their own genomes but requiring many nuclear-encoded proteins.

  • Nucleus: Contains the genetic material; proteins enter via nuclear pores.

  • Plasma Membrane: The cell boundary where integral membrane proteins are embedded.

• The fundamental questions in this field are:

  • How are proteins targeted specifically to these different organelles?

  • What mechanical and biochemical processes occur during this targeting?

• Scale context: A typical cell diagram indicates a scale of approximately $15\,\mu m$.

The Secretory Pathway: Experimental Discovery

• The existence of a defined pathway for secreted proteins was established using Pulse-Chase Experiments in Pancreatic Acinar Cells.

• Experimental Procedure:

  • Pulse: Cells are exposed to a radiolabeled amino acid for a brief period (e.g., a 3-minute label).

  • Chase: The radiolabeled amino acids are replaced with unlabeled ones, allowing researchers to track the "wave" of protein synthesis over time.

• Observations over time:

  • 3-minute label: Radioactivity is concentrated in the Rough Endoplasmic Reticulum (RER).

  • 7-minute chase: Radiolabeled proteins have moved into the Golgi Apparatus.

  • 120-minute chase: Protein is located in Secretory Vesicles near the cell exterior.

• The Definitive Secretory Pathway: ER $\rightarrow$ Golgi $\rightarrow$ Secretory Vesicles $\rightarrow$ Cell exterior.

Intracellular Sorting of Proteins

• Protein synthesis begins on ribosomes in the cytosol. There are two primary populations of ribosomes, which are structurally identical (the same ribosomes move between states):

  • Free Ribosomes in Cytosol: Synthesize proteins that remain in the cytosol or are transported to the Nucleus, Peroxisomes, Mitochondria, or Chloroplasts.

  • Membrane-bound Ribosomes: Synthesize proteins that enter the Secretory Pathway.

• The Secretory Pathway Destinations:

  • Endoplasmic Reticulum (Lumen and Membrane).

  • Golgi Apparatus.

  • Endosomes.

  • Lysosomes.

  • Secretory Vesicles.

  • Plasma Membrane.

  • Extracellular environment (via secretion).

The Endoplasmic Reticulum Signal Sequence (ERSS) and SRP

• Ribosomes determine whether to stay in the cytoplasm or move to the ER surface based on Protein Signals.

• ER Signal Sequence (ERSS):

  • Typically located at the amino (N) terminus of the nascent protein.

  • Characterized by a core of $\ge 12$ hydrophobic amino acids.

  • Contains a cleavage site for signal peptidase.

• Signal Recognition Particle (SRP):

  • Recognizes and binds the signal sequence as it emerges from the ribosome.

  • Composition: A complex consisting of 6 polypeptides and 1 RNA (SRP RNA).

  • Structure: Includes flexible regions designated as Hinge 1 and Hinge 2.

Co-translational Targeting and Translocation

• Step-by-Step Process:

  • Step 1: Ribosome begins translation in the cytosol; the ERSS emerges.

  • Step 2: SRP binds to the ERSS, pausing translation temporarily.

  • Step 3: The SRP-ribosome complex binds to the SRP receptor on the ER membrane.

  • Step 4: The SRP is released, and the ribosome is transferred to the Translocon (a protein-lined channel).

  • Step 5: Translation resumes, and the polypeptide is threaded into the ER lumen through the translocon channel.

  • Step 6: For soluble proteins, signal peptidase cleaves the signal sequence. The protein is released as a free-floating (soluble) molecule into the ER lumen.

Insertion of Integral Membrane Proteins

• Membrane proteins contain hydrophobic segments that anchor them in the bilayer.

• Structural Domains:

  • Extracellular domain: Outside the cell.

  • Transmembrane domain: Crosses the membrane; typically an $\alpha$-helix consisting of $\ge 20$ hydrophobic amino acids.

  • Intracellular domain: Faces the cytoplasm.

• Insertion Mechanisms:

  • Cleavable SS and Stop-transfer sequence: The N-terminal signal sequence starts translocation, but an internal "stop-transfer" hydrophobic sequence ($\sim 20$ AA) halts translocation, causing the protein to be released laterally into the membrane.

  • Internal (Non-cleavable) Signal Sequences: The signal sequence itself acts as a transmembrane domain and is not cleaved. The orientation of the protein (N-terminal vs. C-terminal in the cytosol) depends on the surrounding amino acids.

  • Multipass Proteins: Proteins that span the membrane multiple times (e.g., GPCRs) utilize a series of alternating internal signal sequences and stop-transfer sequences.

Tail-Anchored Proteins and the GET Pathway

• Some proteins have a transmembrane sequence at the very C-terminus, meaning they cannot be targeted co-translationally because it only emerges after translation is finished.

• TRC40 (as GET3 in yeast): A specialized chaperone that recognizes the C-terminal transmembrane domain.

• GET1-GET2 Complex: Acts as an insertase in the ER membrane.

• Mechanism: The TRC40/GET3 factor delivers the protein to the GET1-GET2 receptor. The channel undergoes state changes described as "open channel" (facilitating insertion) and "closed channel" (sealed by GET3/TRC40) to maintain the membrane barrier.

Protein Bioinformatics: Hydrophobicity Plots

• Primary structure analysis allows for the prediction of transmembrane regions.

• Criteria for a Transmembrane Domain:

  • Hydrophobicity value > $+2$ on a hydropathy index.

  • Sequential length > $19$ amino acids.

• Case Study Comparison:

  • Tubulin beta chain (P07437): Highly hydrophilic; corresponds to HP1 (flat plot). Located in the cytosol.

  • Aquaporin-1 (P29972): Contains multiple hydrophobic peaks; corresponds to HP2. Located at the plasma membrane.

Protein Processing and Post-Translational Modifications (PTMs) in the ER

• 1. Proteolytic Processing:

  • Example: Preproinsulin to Insulin.

  • The signal sequence is removed (Signal Peptidase).

  • Disulfide bonds form.

  • The connecting polypeptide (C-peptide) is removed to produce the final active insulin molecule (A and B chains linked by disulfide bonds).

• 2. Formation of Disulfide Bonds:

  • Catalyzed by PDI (Protein Disulfide Isomerase).

  • Environment: The ER lumen is oxidizing (allows $-S-S-$ bonds), whereas the cytosol is reducing (keeps cysteines as $-SH$).

• 3. Folding:

  • BiP: An Hsp70 family chaperone that resides in the ER lumen to assist protein folding and prevent aggregation.

• 4. N-linked Glycosylation:

  • A pre-assembled oligosaccharide is transferred from a dolichol lipid carrier to an Asparagine (N) residue during translocation.

• 5. Addition of GPI Anchors:

  • Proteins destined for the outer cell surface are attached to Glycosylphosphatidylinositol (GPI) anchors.

  • Chemical components: Ethanolamine, Mannose, N-acetylgalactosamine, Glucosamine, Inositol, and Fatty acid side chains.

  • Topology: GPI-anchored proteins face the extracellular environment.

Quality Control and the Unfolded Protein Response (UPR)

• Glycoprotein Folding Sensor:

  • Uses Calnexin and Calreticulin (ER-resident chaperones).

  • Three glucoses initially "stick out" of the N-linked sugar. Two are removed; the remaining one binds to Calnexin.

  • If folding is correct, the final glucose is removed, and the protein proceeds.

  • If folding is incorrect, UDP-Glucosyltransferase adds a glucose back to force another cycle of chaperone binding.

• ER-Associated Degradation (ERAD):

  • If folding fails repeatedly (indicated by the removal of mannose residues), the protein is retro-translocated back to the cytosol.

  • It is then degraded via an Ubiquitin-dependent mechanism.

• Unfolded Protein Response (UPR): Triggered by an accumulation of misfolded proteins. Outcomes include:

  1. General inhibition of protein synthesis.

  2. Increased expression of ER chaperones (BiP, PDI, Calnexin, Calreticulin).

  3. Increased proteasomal activity.

The Golgi Apparatus: Structure and Models

• Organization:

  • ERGIC (ER-Golgi Intermediate Compartment): The sorting station between ER and Golgi.

  • cis Golgi network: Entry face.

  • Golgi stack: Divided into medial and trans cisternae.

  • trans Golgi network (TGN): Exit face for sorting to plasma membrane, secretion, endosomes, or lysosomes.

• Models of Transport:

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

  • Cisternal Maturation Model: The cisternae themselves evolve and move forward, while resident enzymes are recycled backward via vesicles.

• ER Retrieval Sequences:

  • Proteins meant to stay in the ER are retrieved from the ERGIC or Golgi if they escape.

  • KDEL: Retrieval sequence for soluble proteins (at the C-terminus).

  • KKXX: Retrieval sequence for membrane proteins (at the C-terminus).

Golgi Glycosylation and Lysosomal Targeting

• Processing in Golgi:

  • cis: Removal of 4 mannose residues.

  • Medial: Addition of N-acetylglucosamine; removal of 2 additional mannose; addition of fucose and 2 more N-acetylglucosamines.

  • trans & TGN: Addition of 3 galactose and 3 sialic acid residues.

• Lysosomal Targeting:

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

  • They receive a specific tag: Mannose-6-phosphate (M6P) through phosphorylation.

Vesicular Traffic and Transport Mechanisms

• Directionality:

  • Anterograde: Forward traffic (ER $\rightarrow$ Golgi $\rightarrow$ Plasma Membrane).

  • Retrograde: Backward traffic (Golgi $\rightarrow$ ER).

• Coat Proteins (Budding):

  • COP II: Mediates anterograde traffic (ER to Golgi).

  • COP I: Mediates retrograde traffic (Golgi to ER).

  • Clathrin: Mediates traffic between the Golgi, Endosomes, and Plasma Membrane.

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

• Vesicle Fusion:

  • Mediated by SNARE proteins.

  • v-SNARES: Located on the vesicle membrane.

  • t-SNARES: Located on the target compartment membrane.

• Microtubule-Based Movement:

  • Vesicles move along microtubules using motor proteins.

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

  • Dynein: Typically moves toward the minus (-) end (nucleus/center).

Polarized Cells and Sorting

• In specialized cells (e.g., intestinal epithelium), the plasma membrane is divided into Apical and Basolateral domains.

• Tight Junctions: Prevent the mixing of proteins between these domains.

• Protein sorting occurs in the trans-Golgi network or via recycling endosomes.

• Examples of distinct transport pathways:

  • Neurotransmitters in neurons.

  • Digestive enzymes in pancreatic acinar cells.

  • Hormones in endocrine cells.