ER and Golgi Vesicular Transport

Vesicular Transport Overview

• Early to late endosomal transport relies on microtubules.
• Vesicular transport involves the ER, Golgi apparatus, and lysosomes.

ER to Golgi Transport

• Governed by the "Default Pathway," which describes protein movement after import into the ER.
• Proteins generally move along this route: Rough ER -> Golgi -> Plasma membrane via vesicles
• Diverting from this pathway necessitates specific signals, such as retention or diversion signals.
• The Golgi is crucial for biosynthesis, sorting, dispatch, and recycling of proteins.

Golgi Apparatus Features

• Located near the nucleus (perinuclear)

Golgi Stacks

• Consists of cis, medial, and trans regions
• Cis: the first region, closest to the ER, and the point of protein entry.
• Medial: the middle region where proteins undergo processing.
• Trans: the final region where proteins are sorted and directed to their final destinations.
• Function: Modifies glycoproteins by adding or removing sugars, using glycosidases (removes sugars) and glycosyltransferases (adds sugars).
• Cis and trans faces act as sorting centers.
• Cis-Golgi Network: Sends proteins to the cis-Golgi stack or returns them to the ER.
• Trans-Golgi Network: Directs proteins to the plasma membrane, lysosomes, or secretory vesicles.

ER to Golgi Transport and Export

• Secretion is the rate-limiting step in protein transport.
• The ER machinery assesses protein folding and subunit assembly as a quality control step.

ER Quality Control Steps

• If No: Misfolded or unfolded proteins are degraded, which can be detrimental.
• If Still Folding: Protein remains in the ER bound to BiP or other chaperones.
• If Yes: Protein is exported via the default pathway to the cis-Golgi network.

ER Resident Proteins

• They are retained using retention and retrieval mechanisms.
• They have a C-terminal KDEL sequence (Lys-Asp-Glu-Leu) as a retention signal.
• KDEL receptors exist in the ER and the cis-Golgi network.
• If an ER-resident protein escapes to the Golgi, the KDEL receptor sends it back to the ER using transport vesicles.
• Removal of the KDEL sequence leads to transport to the cell surface or extracellular space.
• Fusing KDEL sequences to non-ER proteins retains them in the ER.

KDEL Retrieval Mechanism

• Keeps ER-resident proteins in the ER, even if they escape to the Golgi.
• ER soluble proteins like BiP have a KDEL tag
• ER transmembrane proteins have a KKXX sequence, which interacts with COPI vesicles for retrograde transport.
• Recognition of ER proteins by KDEL receptors needs an acidic environment, which occurs in the Golgi but not the ER.
• Forward pathway: ER -> Golgi and is anterograde transport
• Backward pathway: Golgi -> ER and is retrograde transport
• Retrograde transport uses microtubules and Nocodazole disrupts microtubules, preventing ER protein retrieval.

Protein Folding and Maturation in the Rough ER

• Some ER proteins are resident, while others are en route to other destinations.
• Proteins must be correctly folded, modified, and assembled before exiting the rough ER.
• Chaperones like BiP, calnexin, and calreticulin prevent aggregation, facilitate folding using ATP.
• Isomerases include protein disulfide isomerase (PDI).
• Glycosyltransferases and glycosidases.

N-Glycosylation

• Happens on Asparagine residues (N)
• Almost all proteins in the ER lumen undergo post-translational glycosylation where carbohydrates are added after synthesis.
• Modified proteins are called glycoproteins.
• A complex oligosaccharide is transferred all at once, instead of adding sugars individually.
• Consensus glycosylation sequence: Asn-X-Ser/Thr.

Synthesis of Oligosaccharide Precursor

• Occurs via biosynthesis of GPI-anchored proteins.
• Assembly begins in the cytosol.
• Sugar molecules are linked to nucleotide-lipid carriers
• Key sugar donors in the cytosol include UDP-GlcNAc and GDP-Man.
• These sugars are transferred to a lipid-phosphate carrier sequentially.
• Carrier lipid: Dolichol-Phosphate (Dol-PO₄)
• (Dol-PO₄) is a polyisoprenoid lipid in the ER membrane, which acts as an anchor and transports it across the ER membrane.

Flipping to the ER Lumen

• Once a partial GlcNAc-Man structure assembles, a flippase enzyme moves it into the ER lumen.

Final Modifications in the ER Lumen

• More sugar residues are added using sugar donors: Dol-P-Man and Dol-P-Glc.

Protein Glycosylation Reaction

• The enzyme oligosaccharyltransferase (OST) transfers a pre-made oligosaccharide to the Asn-residue on the target protein.
• OST is closely associated with the SEC61 complex, so protein modifications occur quickly after translocation into the ER lumen.

Glycan Trimming

• The glycoprotein undergoes trimming before exiting the ER: 3 glucose (Glc) residues and 1 mannose (Man) residue are removed.
• Further sugar removal and addition occur in the Golgi.
• Glycosidases remove sugars and glycosyltransferases add sugars.

Sugar Processing in the Golgi

• Complex and high mannose oligosaccharides are generated and modified.

Golgi Structure Maintenance

• The Golgi structure is maintained by two models

Vesicular Transport Model

• Static Golgi with vesicles ferrying cargo between the stacks.

Cisternal Maturation Model

• Golgi cisternae mature moving from the cis to the trans face.
• Retrograde transport brings back the enzymes needed for maturation.

Transport to and From Lysosomes

• Lysosomes degrade and recycle macromolecules.
• Acidity is maintained by V-Type ATPase pumping hydrogen ions into the lysosome.
• They contain numerous acid hydrolases, such as nucleases, proteases, and glycosidases.
• They are electron dense.

Delivery and Trafficking to Lysosomes

• The lysosome receives material from several pathways:
• Biosynthetic pathway: ER → Golgi → Lysosome
• Endocytic Pathway: Uptake of external materials
• Autophagic Pathway: Self-degradation of cellular components
• Phagocytic Pathway: Engulfment of large particles in immune cells like macrophages and neutrophils.

Sorting of Lysosomal Hydrolases

• Lysosomal hydrolases are located and sorted by a two-step signaling system.

Signal Patch & Mannose-6-Phosphate (M6P) Tagging

• Enzymes destined for lysosomes have a signal patch (3D structure) in the cis-Golgi network.
• Glycosyltransferases recognize the signal patch and add an M6P tag.
• The M6P tag acts as a shipping label, directing the enzyme to lysosomes.

Mannose-6-Phosphate (M6P) Receptor Sorting

• M6PRs in the trans-Golgi network (TGN) recognize and bind M6P-tagged lysosomal enzymes.
• Receptors segregate lysosomal enzymes from other proteins for transport to different locations.
• The M6PR-bound enzymes are packaged into transport vesicles that go to late endosomes before delivery to lysosomes.

Lysosome Membrane Protein Pathway

• LAMPs do not receive the M6P tag.
• LAMPs follow the default pathway - Synthesized with the ER, Processed by the Golgi, and Transported to the Plasma membrane
• LAMPs are then endocytosed and trafficked to lysosomes.
• Hydrolases and M6P receptors use this pathway in small amounts
• The scavenger pathway corrects misdirected lysosomal proteins and prevents the loss of essential enzymes.

Lysosomal Storage Diseases

• They are caused by gene mutations.
• Examples include Hurler’s Disease and I-cell disease.
• Hurler’s disease is characterized by missing lysosomal enzymes needed for glycosaminoglycan (GAG) breakdown.
• Co-culturing diseased cells with normal cells rescues the diseased cells.
• Man-6-P receptors bind and internalize enzymes secreted by normal cells.
• I-cell disease is due to a defective GlcNAc phosphotransferase.
• There is no M6P tag, M6P receptors cannot sort and target hydrolases, and hydrolases are missing from lysosomes.

Endocytosis

• It is the vesicular import of particles and macromolecules.

The Need for Endocytosis

• Occurs in the plasma membrane.
• Functions include uptake of nutrient molecules, clearance of harmful substances, and maintenance of surface-to-volume ratio.

Types of Endocytosis

• They are distinguished by the size of the import vesicle and the material being ingested
• Phagocytosis: uptake of large matter (greater than 250 nm)
• Receptor-mediated endocytosis: uptake of macromolecules using specific cell surface receptors (250-300 nm)
• Pinocytosis: uptake of fluid and small solutes (less than 150 nm).
• Most ingested material is delivered to lysosomes.

Phagocytosis

• It is the uptake of large particles using generic receptors
• Practiced by protozoa, macrophages, neutrophils, and fibroblasts.
• It is receptor-mediated.
• Cells bind the particle and extend pseudopodia to engulf it.
• Engulfment requires receptor-ligand interactions around the surface.

Extension of Pseudopodia

• Receptor-ligand interactions induce changes in cortical cytoplasm.
• Calcium fluxes induce the formation of gel-like cytoplasm.
• Actin filaments polymerize, pushing the membrane outward to engulf the target.
• Once the pseudopodia fuse, the particle is inside a vesicle called a phagosome.

Degradation of Ingested Particles

• Particles enter reside in phagosomes.
• Oxygen metabolites kill bacteria as the phagosome undergoes an oxidative burst.
• Lysosomes fuse with phagosomes to form phagolysosomes which are acidic due to low pH and activates acid hydrolases which helps to digest particles
• Residual material remains in the phagolysosome as a residual body.

Virulence Mechanisms Used by Pathogens

• Bacteria secretes hemolysin and undergoes actin polymerization.
• Agents use various mechanisms to infect hosts and avoid the immune system.
• Some pathogens escape the phagosome using the Escape mechanism
• Type 1 escape mechanism: Bacteria has hemalysin secretion, for host release to replicate
• Type 2 escape mechanism (using host machinery): Bacteria is engulfed with the Zipper mechansim
• Some pathogens survive in the phagolysosome instead
• Zipper Mechanism enables bacterial proteins to bind to specific receptors for engulfment.
• Trigger Mechanism involves injecting proteins that induce cytoskeletal rearrangements.

Receptor-Mediated Endocytosis

• Selective form where membrane receptors bind a ligand
• Vesicular uptake of macrocolecules, nutrients, iron, growth factors, modified glycoconjugates all included
• Cells bound by receptors increase uptake 1000x
• Receptor-ligand entercells via clathrin-coated plaques

Clathrin-Coated Pits

• specialized structures collecting receptors

Clathrin Assembly

• Drive formation of vesicle
• Composed of heavy and light chains with adapator complexes for interaction

Clathrin Adaptators

• Mediate clathrin binding- 2 types, plasma and trans-golgi
• Heterotetramers

Clathrin signal motif

• Endocytic recpetors have a 4 amino acid motif
• Key feature is protruding loop

Clathrin Cycle

• Form, bud, uncoat, and reuse vesicles

Endosomal Compartments

• Formed through maturation, microtubule transports
• pH in vesicles decrease due to the pathway
• Early sorting, lysosome late endosome

LDL mediated pathway for receptors

• Bind with receptors, uncoat the clathrin, and fuse with endosome
• then ligands are recycled

Cellular Response to Insulin

• unstimulated has no insulin, has transporters inside cells
• Glucose uptake is limited
• Stimulated = insulin binds and starts cascade
• Increases glucose amount in the the blood

Late Endosomes

Sort receptors for recycling

• Release Cholesterol Have lysosomes eventually

MVB pathway

• Proteins degraded are tagged by monoubiquitin This ubiquitin tag is recognized by complex ESCRT Sequestered into the MVBs for transport to cells - recylced
• Important for growth cells

Niemann Pick Diseases

• Bad endosomes cause lipid accumaliation
• Lipid kills spleen/ liver- neurologcial issue- type A- fatal

NPC disorder

• Issue = NPC protein transport with lipid
• leads to bad trafficking and retrorgrade transport

Transytosis- transfers cargo

• The cell transports material across to membrane (apical and basal )
• recptors and ligands recycled

Pinocytosis

• Fluid uptake
• Maintenace of protein turnover
• Occurs with or whitout clathrin

Caveolae

• small indentation with caveolin protein
• enriched with signalmolecules

Exocytosis/secretions

Transport the cargo and balances between exo and endos

• unregulated continous transport
• Signaldependant secretion

Regulated Secreation

• Specialized for cells neurotransmitters
• Transports packages

Vesical membrane pathway

Once its triggered, release via molecular signals and moves

Key concepts for docking

• ligand surface - signalling
• increase clamcium
• disruptive cytoskel