• Nuclear Localization Sequences ( NLS ) :

  • Imports = basic : lysine ( K ) , arginine ( R ) , histidine ( H )

  • Exports = aliphatic : isoleucine ( L )

    • LxxxLxxLxL

• Nuclear Import Process :

  1. cargo-NLS is complexed with an importin

  2. the complex travels through the Nuclear Pore Complex ( NPC ) from the cytosol , into the nucleus

  3. Ran-GTP binds to the complex , allowing the cargo to be released from the importin

  4. Ran-GTP bound to the important exits the nucleus back into the cytosol

  5. Ran-GTP is hydrolyzed into Ran-GDP and the importin is released

• Nuclear Localization Signals use the adapters like importins to facilitate movement into the nucleus

• Mitochondria also use signal localization sequences

  • instead of importins , they use TOM / TIM pore complexes

  • the localization sequence gets cleaved once its inside

  • depends on heat shock proteins as chaperones

• Peroxisomes also use short signal sequences to get proteins from ER ➡️ peroxisome

• Signal sequences are also used to direct partially started ribosomal complexes to the ER wall itself

  • here it can stabilize , and continue translation , dumping the contents into the ER lumen

  • N-terminal domain enters into the ER lumen first

• Glycosylation is very common for rough ER synthesized proteins

  • the tags mark the state of protein folding

• Improperly folded proteins get ubiquitinated , and then exported into the cytosol , to the be broken down in peroxisomes

• Accumulation of Improperly folded proteins ,

  • triggers gene transcription of chaperones to assist in folding

• Vesicle Coatings :

  1. Clathrin = normal endocytosis

  2. COPI = golgi ➡️ ER

     • retrograde , backward direction

  3. COPII = ER ➡️ golgi

     • anterograde , forward direction , for export

• Dynamin = GTPase that pinches off vesicles

  • uncoats clathrin

• Rab = used to guide endosomes cargo to its target location in the cell

• SNARES mediate membrane fusion

  • they need to be separated after

  • used in exocytosis

• Endocytosis :

  • clathrin = normal

    • Process : Cargo binds to receptors, adaptors recruit clathrin, and vesicle formation begins. Dynamin constricts the vesicle neck, enabling release.

    • Uncoating : ATP-driven disassembly of the clathrin coat for vesicle integration with endosomes.

  • pinocytosis = water

  • LDL fats = also use clathrin

• Exocytosis :

  • Continuous : Supplies membrane and secretory proteins without regulation

  • Stimulus-Dependent : Triggered by external signals, often involving calcium influx binding to synaptotagmin, facilitating vesicle fusion with the plasma membrane.

  • SNARE Complex :

    • Components: Synaptobrevin ( V-SNARE ) , Syntaxin , and SNAP-25 ( T-SNAREs )

• Parkinson's Disease :

  • can't release or reuptake dopamine

  • Parkin 1 = accumulates and prevents synaptic vesicle release

  • 𝛼-synuclein = blocks SNARE complexes , aka vesicle fusion

• Golgi = sorting device :

  1. lysosomes

  2. secretory vesicles ( signal mediated )

  3. secretory vesicles ( constitutive secretion )

• Pathways to a Lysosome :

  • enocytose , phagocytose , micropinocytose , or autophagy of mitochondrion

• Mannose-6-Phosphate Receptors = on golgi , used in early endosome formation

• Neurons :

  • golgi ➡️ synaptic vesicle ( endosome ) ➡️ fuses , offloads contents into synaptic cleft ➡️ contents "re-uptook" / endocytosed back into pre-synaptic terminal

Recognizing Signaling Sequences for Protein Sorting

• Function : Signal sequences are short amino acid sequences that function as 'zip codes' to direct proteins to their proper cellular locations.

• Examples :

  • Nuclear Localization Signals ( NLS ) : Directs proteins to the nucleus.

  • Mitochondrial Targeting Sequences ( MTS ) : Guides precursor proteins to the mitochondria.

  • Endoplasmic Reticulum ( ER ) Signal Peptides : Target nascent polypeptides to the ER membrane.

• Imports = typically , an NLS consists of one or more short stretches of positively charged amino acids, such as lysines ( K ) and arginines ( R )

  • It can appear as a continuous sequence or in bipartite form ( two basic clusters separated by a few amino acids )

  • Example = PKKKRKV

• Exports = NES sequences are often characterized by a pattern of hydrophobic amino acids, commonly leucine-rich stretches

  • A canonical NES might look like LxxxLxxLxL , where "L" represents leucines and "x" represents any amino acid

Nuclear Pore Complex ( NPC )

• they perforate the nuclear envelope

  • large , complex structure embedded in the nuclear envelope

• act as gatekeepers of the nuclear envelope.

  • selective gateway for macromolecular traffic

Movement of Proteins Across the Nuclear Pore Complex

• Import Mechanism ( NLS Cargo Proteins ) :

  1. Binding: Cargo proteins with an NLS bind to importins in the cytoplasm.

  2. Transport: The importin-cargo complex translocates through the nuclear pore complex ( NPC ).

  3. Release: In the nucleus, Ran-GTP binds to importins, causing a conformational change that releases the cargo protein.

  4. Recycling: The importin bound to Ran-GTP is transported back to the cytoplasm, where Ran-GAP triggers the hydrolysis of Ran-GTP to Ran-GDP, allowing the importin to be reused.

• Export Mechanism ( NES Cargo Proteins ) :

  1. Binding: Cargo proteins with an NES bind to exportins in the nucleus, forming a complex with Ran-GTP.

  2. Transport: The exportin-cargo-Ran-GTP complex translocates through the NPC into the cytoplasm.

  3. Release: In the cytoplasm, Ran-GAP facilitates the hydrolysis of Ran-GTP to Ran-GDP, leading to the release of the cargo protein and exportin.

  4. Recycling: The exportin and Ran-GDP return to the nucleus for reuse, with Ran-GDP being converted back to Ran-GTP by Ran-GEF.

• The Role of Ran in Directionality :

  • The NLS is crucial for recognizing proteins that need to be imported into the nucleus

  • The NES tags proteins that need to be exported.

  • The Ran gradient ensures that the binding and release of cargo by importins and exportins occur in the correct compartments ,

    • maintaining the directionality and efficiency of transport

  • Ran-GTP binding within the nucleus induces cargo release

  • Ran-GTP hydrolysis in the cytoplasm resets the receptor

  • directionality is imposed by the gradient of Ran-GTP and Ran-GDP

  • Ran-GDP ( inactive form ) : Predominantly located in the cytosol

  • Ran-GTP ( active form ) : Concentrated in the nucleus

Mitochondrial Protein Import

• Signal Sequences: Mitochondrial proteins are tagged with N-terminal presequences that are recognized by mitochondrial translocases.

• Key Translocases:

  • TOM Complex ( Translocase of the Outer Membrane ) : Initial entry site for precursor proteins.

  • TIM Complexes ( Translocase of the Inner Membrane ) : Facilitate transport across the inner membrane or into the mitochondrial matrix.

• Energy Requirements:

  • ATP Hydrolysis: Required for chaperones assisting protein translocation.

  • Membrane Potential: Drives precursor proteins through the inner membrane.

• Folding Mechanisms:

  • Proteins must remain unfolded during translocation, mediated by cytosolic and mitochondrial chaperones.

Protein Import into Peroxisomes

• A short Signal Sequence Directs the Import of Proteins into Peroxisomes

• ER ➡️ Peroxisome Precursors ( empty )

  • signal recognition sequence on unfolded protein allows entry , forming mature peroxisome

Unfolded Protein Response ( UPR ) in the ER

• UPR = includes an increased transcription of genes encoding proteins involved in retrotranslocation and protein degradation in the cytosol, ER chaperones, and many other proteins that help to increase the protein-folding capacity of the ER.

• Trigger: Accumulation of misfolded proteins in the ER lumen.

• Outcome: Enhanced transcription of chaperones and folding enzymes to manage ER stress. Failure to restore homeostasis can trigger apoptosis.

• Mechanism :

  1. Misfolded Proteins Sensors ( IRE1 , PERK , ATF6 ) detect high amount of Misfolded Proteins in the ER

  2. Create Transcription Factors

  3. Activation of Genes that aid in Protein Folding ( chaperones )

• Signal Sequences were First Discovered in Proteins Imported into the Rough ER

• A Signal Recognition Particle Directs the ER Signal Sequence to a Specific Receptor

• Most Proteins Synthesized in the Rough ER are Glycosylated by the addition of a common N-linked Oligosacchararide

• Oligosaccharides are used as Tags to Mark the State of Protein Folding

• Improperly Folded Proteins are Exported from the ER and Degraded in the Cytosol

• Translocation across ER membrane does not always require ongoing polypeptide chain elongation

• In Single-Pass Transmembrane Proteins, a Single Internal ER Signal Sequence Remains in the Lipid Bilayer as a Membrane spanning alpha Helix

• The UPR is a cellular stress response related to the endoplasmic reticulum (ER).

• When misfolded proteins accumulate, signaling pathways are activated to enhance protein-folding capacity, degrade misfolded proteins, and reduce general protein synthesis to restore homeostasis.

• If unsuccessful, apoptosis may be triggered.

Types of Coated Vesicles :

1. Clathrin-Coated Vesicles:

   • Function: Mediate endocytosis from the plasma membrane to endosomes and transport between the Golgi and endosomes.

   • Adaptor Proteins: Link cargo receptors to clathrin, facilitating vesicle formation.

2. COPI-Coated Vesicles:

   • Function: Retrograde transport from the Golgi apparatus back to the ER.

3. COPII-Coated Vesicles:

   • Function: Anterograde transport from the ER to the Golgi.

Vesicle Formation and Uncoating :

• Adaptor Proteins: Recognize and bind cargo, linking them to coat proteins like clathrin.

• Dynamin: A GTPase that pinches off vesicles from donor membranes through GTP hydrolysis.

• Rab GTPases: Guide vesicles to their target membranes by tethering vesicles to effector proteins.

• SNARE Proteins: Mediate fusion by forming a complex between v-SNAREs on the vesicle and t-SNAREs on the target membrane, facilitating membrane merging.

Endocytosis:

• Clathrin-Mediated :

  • Process : Cargo binds to receptors, adaptors recruit clathrin, and vesicle formation begins. Dynamin constricts the vesicle neck, enabling release.

  • Uncoating : ATP-driven disassembly of the clathrin coat for vesicle integration with endosomes.

• Pinocytosis : Non-clathrin-mediated uptake, common for fluid-phase endocytosis.

• LDL Receptor-Mediated : Utilizes clathrin-coated pits for cholesterol transport.

Exocytosis :

• Constitutive Pathway :

  • Continuous: Supplies membrane and secretory proteins without regulation.

• Regulated Pathway :

  • Stimulus-Dependent : Triggered by external signals, often involving calcium influx binding to synaptotagmin, facilitating vesicle fusion with the plasma membrane.

• SNARE Complex :

  • Components: Synaptobrevin ( V-SNARE ) , Syntaxin , and SNAP-25 ( T-SNAREs )

  • Regulation: Complexin stabilizes the partially assembled SNARE complex, while calcium binding to synaptotagmin catalyzes membrane fusion.

Golgi Apparatus Function and Protein Sorting

• Compartmentalization: The Golgi is composed of cis , medial , and trans compartments

• Processing:

  • N-linked Glycosylation: Modified further in the Golgi.

  • Post-translational Modifications: Includes phosphorylation, sulfation, and addition of sugar moieties.

• Sorting Pathways:

  1. Signal-Mediated Diversion to Lysosomes: Involves mannose-6-phosphate tagging and receptor recognition.

  2. Regulated Secretory Pathway: Stores proteins for release upon stimulation.

  3. Constitutive Pathway: Continuous secretion of cellular proteins.

Transport from the ER to the Golgi and Retrieval Pathways

• COPII-Coated Vesicles: Facilitate transport from the ER to the Golgi.

• Vesicular Tubular Clusters: Form intermediate structures transported along microtubules.

• Retrieval Pathway:

  • COPI-Coated Vesicles: Return ER-resident proteins marked with KDEL sequences to the ER, maintaining cellular protein homeostasis.

Golgi

• The Golgi Sorts Between ( Trans Golgi Network ) :

  1. signal-mediated diversion to lysosomes ( via endosomes )

  2. signal-mediated diversion to secretory vesicles ( for regulated secretion )

  3. constitutive secretory pathway

Parkinsons

• Parkin 1 = prevents synaptic vesicle release

  • ubiquinates Synaptotagmin

• Pink 1 = associated with mitochondrial quality control, impacts vesicle recycling.

• 𝛼-synuclein = blocks vesicle fusion by blocking SNARE complex

• Outcome: Dysregulated dopamine release and reuptake, leading to potential dopaminergic neuron toxicity.

Misc

• There are Various Types of Coated Vesicles

• Adaptor Proteins Select Cargo into Clathrin Coated Vesicles

• Dynamin Regulates the Pinching-off and Uncoating of Clathrin Coated Vesicles

• Rab Proteins Guide Transport Vesicles to their Target Membrane

• Rab protein Guidance to Target Membrane

• SNAREs Mediate Membrane Fusion

  • V-SNARE and T-SNARE

  • Calcium entry into cell binds to synaptotagmin, resulting in fusion of membranes

  • SNAREs need to be Separated after Fusion

• Golgi Apparatus Consists of an Ordered Series of Compartments

• Pathways to lysosomes

  • endocytose something ➡️ lysosome

  • phagocytose something ➡️ lysosome

  • macropinocytosis ➡️ lysosome

  • autophagy of mitochondria ➡️ lysosome

• Manose 6-phosphate Receptor

  • on golgi

  • on early endosomes

• Pinocytosis : no clathrin coat

• LDL uses clathrin mediated endocytosis