cell lecture 7 (m2)

Cellular Trafficking I

Protein Sorting

• Newly synthesized proteins post-translation can be targeted to different cell compartments

Processes of Protein Sorting

• Co-translational Import:

  • Occurs during translation into the endomembrane system.

• Post-translational Import:

  • Takes place after translation into other organelles.

Endomembrane System

Function and Components

• The endomembrane system is critical for:

  • The endomembrane system is a network of membrane-bound organelles involved in the synthesis, modification, transport, and degradation of proteins and lipids.

    Endomembrane Components:

  • Nuclear Envelope – Surrounds the nucleus and connects with the rough ER.

  • Endoplasmic Reticulum (ER)

    • Rough ER: Site of protein synthesis (has ribosomes).

    • Smooth ER: Site of lipid and steroid synthesis, lacks ribosomes.

    • Transitional ER: Helps form transport vesicles that carry proteins and lipids to the Golgi.

  • Golgi Complex – Modifies, sorts, and packages proteins & lipids for their final destinations.

  • Endosomes – Sort and traffic material imported into the cell (e.g., nutrients, signaling molecules).

  • Lysosomes – Break down waste, foreign material, and old organelles using digestive enzymes.

  • Transport Vesicles – Carry molecules between organelles (e.g., from the ER to the Golgi).

  • Plasma Membrane – Receives vesicles from the Golgi, allowing materials to be secreted from the cell.

Rough Endoplasmic Reticulum (ER) (first step of the endomembrane pathway)

Structure and Function

• Rough ER characteristics:

  • The rough ER consists of large, flattened membrane sheets that are continuous with the outer nuclear membrane.

  • Its surface is covered in ribosomes, which make it look "rough" under the microscope.

  • It is involved in protein synthesis, processing, and modification.

Rough ER and Protein Synthesis

• Proteins destined for secretion or for the endomembrane system begin their synthesis on free cytosolic ribosomes.

• These proteins contain an N-terminal ER signal sequence, which directs them to the rough ER.

• A signal recognition particle (SRP) binds to the signal sequence, pausing translation.

• The SRP binds to an SRP receptor on the ER membrane, docking the ribosome to the translocon complex (including Sec61 and signal peptidase).

• GTP hydrolysis releases SRP, and the polypeptide is inserted into the ER lumen through the translocon.

• The signal sequence is cleaved by signal peptidase, and translation continues inside the ER.

Initial Glycosylation:

• The first steps of N-glycosylation occur in the rough ER.

• A lipid-linked oligosaccharide is transferred to the asparagine (Asn) residue of the growing polypeptide chain.

• This aids proper protein folding.

• The remainder of glycosylation and modifications occur in the Golgi apparatus.

Protein Folding and Quality Control in Rough ER

• Two key proteins, calnexin (CNX) and calreticulin (CRT), act as chaperones to assist in proper folding.

• These interact with protein disulfide isomerases (PDI), which help form disulfide bonds.

• Glucosidase enzymes (Glucosidase I and II) remove terminal glucose residues.

• CNX and CRT bind to monoglucosylated proteins, attempting to correct misfolded proteins.

• If a protein fails to fold correctly, UDP-glucose:glycoprotein glucosyltransferase (UGGT) re-adds a glucose molecule, allowing CNX/CRT to attempt refolding.

• If the protein still misfolds, ER Mannosidase I removes mannose residues, signaling it for degradation.

• The misfolded protein is ejected from the ER and degraded by proteasomes via the ER-associated degradation (ERAD) pathway.

Smooth Endoplasmic Reticulum (SER)

Structure and Functions

• Smooth ER features:

  • Lumen is continuous with the rough ER.

  • Tubular shape (compared to the flattened sheets of rough ER).

  • No ribosomes, meaning it does not participate in protein synthesis.

  • Prominent in cells that synthesize steroid hormones (e.g., adrenal glands, testes, ovaries).

Major Functions of the Smooth ER

1. Steroid & Lipid Biosynthesis

• The smooth ER is the primary site of lipid and steroid biosynthesis.

• Cells involved in cholesterol & steroid hormone synthesis have large amounts of smooth ER.

• Enzymes involved in sterol biosynthesis are localized to the SER.

  • HMG-CoA reductase: The rate-limiting enzyme in cholesterol biosynthesis.

  • Target of statin drugs, which lower cholesterol by inhibiting this enzyme.

2. Drug Detoxification in Liver Cells

• Smooth ER in hepatocytes (liver cells) contains Cytochrome P450 (CYP) enzymes, which:

  • Modify and solubilize biotics/xenobiotics (drugs and toxins).

  • ~57 CYP enzymes carry out 75% of drug metabolism in humans.

  • Hydroxylation of hydrophobic drugs/toxins increases their solubility, allowing excretion in bile.

• Repeated drug use induces CYP enzymes, increasing smooth ER production → drug tolerance.

3. Carbohydrate Metabolism – Glycogen Breakdown

• Smooth ER in liver hepatocytes helps regulate blood glucose.

• Breaks down glycogen, which forms dense granules in liver cells.

• Contains glucose-6-phosphatase, an enzyme that:

  • Converts glucose-6-phosphate into free glucose.

  • Releases glucose into the bloodstream during fasting.

4. Calcium Storage (Sarcoplasmic Reticulum)

• Specialized smooth ER in muscle cells = Sarcoplasmic Reticulum (SR).

• Stores & releases Ca²⁺ to regulate muscle contraction.

• Critical for calcium signaling in various cell types.

Membrane Biosynthesis & Lipid Transport

• The smooth ER synthesizes phospholipids for:

  • The plasma membrane.

  • The Golgi & other organelles.

• Phospholipid translocators (flippases) establish membrane asymmetry.

• Membrane transport to other organelles occurs via vesicles.

• Lipids are transferred to mitochondria, peroxisomes, and chloroplasts using phospholipid exchange proteins.

ER Contact Sites with Other Organelles

• Mitochondria-Associated Membranes (MAMs):

  • Specialized smooth ER subdomains that facilitate lipid & calcium exchange with mitochondria.

  • Dysfunction in MAMs is linked to ALS (Lou Gehrig’s disease).

• WrappER (rough ER-mitochondrial association):

  • Found predominantly in liver cells.

  • Involved in lipid transfer & metabolic regulation.

Golgi Apparatus

Structure and Function

• Flattened, disc-shaped membrane sacs called cisternae, stacked together.

• Polarized organelle, meaning different regions have distinct functions.

• Functionally & physically linked to the ER through vesicle trafficking.

Three Main Regions of the Golgi

1. Cis-Golgi Network (CGN)

   • Faces the ER.

   • Receives vesicles containing newly synthesized proteins and lipids.

   • Begins sorting proteins based on their final destination.

2. Medial Cisternae

   • Middle region where protein modifications occur.

   • Adds, removes, or modifies sugars and functional groups.

3. Trans-Golgi Network (TGN)

   • Faces the plasma membrane.

   • Final packaging & sorting of materials into vesicles.

   • Ships vesicles to plasma membrane, lysosomes, or other organelles.

Transport Through the Golgi

• Materials move through the Golgi from the cis side to the trans side.

• Two main trafficking directions:

  • Anterograde movement (ER → Golgi → Plasma membrane)

    • Moves newly synthesized proteins and lipids toward their final destinations.

  • Retrograde movement (Plasma membrane → Golgi → ER)

    • Returns enzymes, misrouted proteins, and excess lipids back for reuse or recycling.

Cisternal Maturation Model – How the Golgi Works

• Cisternae "mature" as they move from the cis-Golgi to the trans-Golgi.

• Proteins stay inside the cisternae, while enzymes remain in specific regions.

• As the cisternae migrate, they undergo enzymatic modifications until reaching the trans-Golgi.

• By the time proteins reach the trans-Golgi, they are fully modified and sorted for transport.

Golgi Functions

Glycosylation – Modification of Proteins & Lipids

• Glycosylation = Addition of carbohydrate chains to proteins and lipids.

• Occurs in both the ER and Golgi, but the Golgi refines and completes modifications.

• Biochemical Polarity:

  • Different Golgi compartments contain specific enzymes for modification.

  • This ensures each protein is processed correctly before reaching its destination.

Types of Glycosylation in the Golgi

1. N-linked glycosylation (Asparagine-linked)

   • Begins in the ER, continues in the Golgi.

   • Adds oligosaccharide chains to the nitrogen (N) of asparagine residues.

   • Further modified in the Golgi for functional targeting (e.g., lysosomal proteins).

2. O-linked glycosylation (Serine/Threonine-linked)

   • Occurs only in the Golgi.

   • Adds sugars to the oxygen (O) of serine/threonine residues.

   • Plays a role in cell signaling and surface recognition.

Modifications at Different Golgi Regions

• Cis-Golgi:

  • Initial modification, such as phosphorylation of lysosomal proteins.

• Medial-Golgi:

  • Mannose removal, addition of N-acetylglucosamine.

• Trans-Golgi:

  • Addition of galactose, sialic acid, and sulfate groups.

  • Final processing before protein sorting & vesicle formation.

Secretion – Exporting Proteins & Lipids

• The trans-Golgi Network (TGN) buds off vesicles, sending processed proteins and lipids to their final destinations.

• Two types of secretion pathways:

1. Constitutive Secretion (Always Active)

• Vesicles move directly to the plasma membrane.

• Fuse immediately, releasing contents outside the cell.

• Always “on,” not regulated.

• Example: Mucus secretion in the intestines.

2. Regulated Secretion (Triggered by Signals)

• Vesicles accumulate in the cytoplasm and wait for a signal.

• Triggered by specific cellular signals (e.g., calcium release, hormone binding).

• Example: Neurotransmitter release in nerve cells.

  • Membrane depolarization triggers vesicles to release their contents via exocytosis.

Overview of Protein Sorting

• Proteins are synthesized in the cytoplasm by ribosomes.

• They can be directed to different cell compartments based on their final destination.

• The sorting process can occur either co-translationally (during synthesis) or post-translationally (after synthesis is complete).

• Once a protein reaches its correct location, it generally remains there.

What Are Protein Sorting Tags?

• Sorting tags are short amino acid sequences that determine where a protein stays or where it needs to go after synthesis.

• These tags ensure proteins reach their correct cellular compartment and stay there if necessary.

• Sorting occurs via two pathways:

  • Co-translational sorting (ER-mediated pathway)

  • Post-translational sorting (for cytoplasmic organelles like the nucleus, mitochondria, etc.)

ER Retention and Retrieval Tags

Some proteins must remain in the ER, while others might accidentally leave and need to be retrieved from the Golgi.

A. ER Retention Tags

• Function: Prevent proteins from leaving the ER.

• Tag Name: RXR Sequence (Arg-X-Arg)

  • X = any amino acid.

  • This tag signals that a protein should stay in the ER instead of being transported to the Golgi.

  • Example: ER-resident proteins like chaperones (e.g., BiP) and enzymes involved in protein folding.

B. ER Retrieval Tags

• Function: Bring proteins back to the ER if they accidentally escape to the Golgi.

• Tag Name: KDEL Sequence (Lys-Asp-Glu-Leu)

  • KDEL is found at the C-terminus of ER-resident proteins.

  • Proteins with this tag that reach the Golgi bind to KDEL receptors, which send them back to the ER via retrograde transport.

  • Example: Soluble ER proteins like BiP and PDI (protein disulfide isomerase).

• Additional Retrieval Tag: KKXX (Lys-Lys-X-X)

  • Found on membrane-bound ER proteins (instead of soluble proteins).

  • Works similarly to KDEL but applies to membrane proteins instead of soluble proteins.

How Retrieval Works: The Retrograde Transport Pathway

1. Some ER proteins accidentally escape and enter the Golgi apparatus.

2. Golgi KDEL receptors recognize proteins with the KDEL retrieval tag.

3. The proteins are packaged into transport vesicles for retrograde transport.

4. They are sent back to the ER, ensuring proper localization.

Experimental Evidence for Retrieval Tags

Green Fluorescent Protein (GFP) Fusion Experiments

• Scientists use GFP-tagged proteins to study protein localization.

• Example: GFP fused to a protein containing the HDEL retrieval tag.

  • If the protein is found in the ER, it confirms HDEL functions as a retrieval signal.

Comparison with ER Resident Protein BiP

• BiP (Binding Immunoglobulin Protein) is a well-known ER-resident protein.

• By comparing GFP-tagged proteins with BiP, researchers can determine if a protein remains in the ER.

• Removing HDEL means the protein is NOT retrieved to the ER.

• Having HDEL is necessary AND sufficient for ER retrieval.

Golgi Sorting and Membrane Thickness

Proteins are also sorted in the Golgi based on:

1. Protein Tags (specific sequences that dictate destination)

2. Membrane Thickness

   • Membrane thickness increases from 5 nm (ER) to 8-10 nm (plasma membrane).

   • Short transmembrane domains (TMDs) → Stay in the ER.

   • Longer TMDs → Sorted to the Golgi or plasma membrane.

   • Proteins remain in compartments with similar membrane thickness.

Sorting by Membrane Thickness

• Proteins with shorter TMDs → Stay in ER

• Proteins with longer TMDs → Move toward the Golgi or plasma membrane