PHGY M2

Endoplasmic Reticulum

• Acts as a highway and warehouse.

• Involved in protein translation and production of membrane phospholipids.

Transport Vesicles

• Transport materials between endomembrane organelles.

Endosomes

Involved in endocytosis to bring materials into the cell.

Golgi Apparatus

• Acts as a post office.

• Labels and sorts proteins with signals to direct them towards their final destination.

Lysosome

Involved in waste disposal and macromolecule breakdown.

Peroxisome

• Involved in breakdown of molecules that generate hydrogen peroxide.

• Prevents cell damage.

COPI

• Proteins that need to be returned to the ER use COPI to coat vesicles that are then sent back to the ER.

• COPI shuttles proteins back to the ER from other locations in the cell.

COPII

• COPII shuttles proteins from the ER to the Golgi.

Clathrin

• Clathrin is used by the Golgi to form vesicles for exocytosis, endocytosis, and transport to the endosome.

• Clathrin is used to shuttle vesicles between the trans Golgi network and other destinations, including the plasma membrane for exocytosis and endocytosis from the plasma membrane to other destinations in the cell.

FUNCTIONS OF THE NUCLEUS

1. Allow DNA to be replicated and transcribed into mRNA as needed

2. Regulate which molecules can access the DNA and separate the DNA from other cell compartments. 

3. Keep the DNA organized. DNA is fragile and easily damaged, so any problems with DNA will lead to major problems in the cell and body.

5 components of the nucleus

1. Nuclear envelope

2. Chromatin and Chromosomes

3. Nuclear pores

4. Nucleolus

5. Nucleoplasm and nuclear matrix

Nuclear envelope

1. Controls what molecules have access to the nucleus and separates the nucleus from other cell compartments

2. It is a double membraned structure that encloses the nuclear material

3. The outer membrane is connected to the endoplasmic reticulum which is important for making proteins

Chromatin

1. a complex of DNA and proteins forming highly organized fibres and is located in different defined areas of the nucleus. Some areas will be actively transcribed into mRNA.

Chromosomes

1. highly condensed chromatin found in the nucleus only during cell division.

Nucleolus

1.  creates ribosomal RNAs (rRNA) and assembles them into the ribosomal subunits. 

2. the site of high amounts of rRNA gene transcription, and the DNA that encodes these genes is organized here.

Nucleoplasm

1. a viscous, water-based fluid that is enclosed in the nuclear envelope. The main function of the nucleoplasm is to serve as a suspension substance for the nuclear contents. It contains dissolved molecules and ions that are essential for the functions of the nucleus.

Nuclear Matrix

Network of filaments within the nucleoplasm that helps to organize chromosomes into compartments and provides the scaffold to maintain the shape and structure of the nucleus.

1. Nuclear pore complexes (NPCs)

regulate the molecular (large molecules) traffic coming in and out of the nucleus

Levels of DNA packaging

Level 1: DNA double helix

Level 2: Nucleosomes

Level 3: Chromatin Fibres

Level 4: chromatin looped domains

Level 5: heterochromatin

Level 2: Nucleosomes

• a complex created from DNA being wrapped twice around the protein histones. 

• This shortens the DNA seven-fold

  • Eight core histones form a nucleosome (an octamer)

  • Histone H1 (a protein) pins core DNA (200 base pairs that are wrapped around the core histones) to the octamer

  • Linker DNA is DNA that connects each nucleosomes to the next 

LEVEL 3: Chromatin Fibre:

The string of nucleosomes coiled into a spiraling fibre, called a chromatic fibre. This forms a helical structure with a diameter of approximately 30 to 40 nm. This shortens the DNA 42-fold.

LEVEL 4: Chromatin Looped Domains:

The 30-40 nm chromatin fibre made of nucleosomes is formed into loops with an average length of 300 nm. This shortens the DNA 750-fold.

LEVEL 5: Heterochromatin:

• DNA is folded and compressed even more, into lengths of 700nm

  • Heterochromatin is compressed further into chromosomes during cell division.

EUCHROMATIN VS HETEROCHROMATIN:

Levels 1 to 4 of DNA packaging are called euchromatin. At these levels, DNA is active, meaning that it can be easily accessed by proteins responsible for: 

• Replicating the chromosomes. 

• Reading a strand of DNA to make RNA. 

Level 5 of DNA organization is called heterochromatin. At this level, DNA is condensed beyond loop domains and is rendered essentially inactive.

Exocytosis

the process of moving cargo out of the cell using the the exocytic pathway.

Endocytosis

• the process of transporting cargo into the cell using the endocytic pathway.

Exocytic Pathway

The process by which a cell directs the contents of secretory vesicles to the plasma membrane and comprises the endoplasmic reticulum, Golgi apparatus, and exocytic vesicles.

Endocytic Pathway

 A process by which cells absorb molecules by engulfing them comprising early and late endosomes and lysosomes.

Parts of the endomembrane system

• rough ER

• smooth ER

• Golgi apparatus

• nucleus

• transport vesicles

Rough Endoplasmic Reticulum

• Covered in ribosomes which give it a ‘rough’ appearance

• Protein translation occurs in the ribosomes and modifications occur here

Smooth Endoplasmic Reticulum

• Is not covered in ribosomes and has a ‘smooth’ appearance

• Processes lipids, metabolizes carbohydrates and is able to store calcium (regulate calcium ions in muscle cells)

Vesicle mediated transport

• bud off from lipid membranes of organelles or the cell as a whole, with the purpose of transporting cargo, such as soluble or membrane-bound proteins. 

• can transport cargo into or out of the cell, or within the cell.

• Once reaching the final destination, they fuse with other lipid membranes to deposit cargo.

Proteins targeted to the endomembrane system:

• Targeted to the ER, golgi complex, lysosomes, the plasma membrane or other organelles contain target signal sequences or are tagged within the endomembrane system

• The proteins end up in various places as transmembrane proteins or in the extracellular space depending on how they were tagged

Proteins destined for the cytosol:

• These proteins lack signal peptides, since free-floating ribosomes will transfer them in the cytosol. Thus, a target signal sequence is not needed

Parts of the golgi apparatus

• Cis Golgi Network

  • Proteins that have entered the endomembrane pathways are received here

• Medial Golgi Network

  • Oligosaccharides can be modified here and added onto proteins

  • Sugar groups can also be added onto lipids

• Trans Golgi Network

  • Materials are sent to specific organelle destinations 

    • Some proteins have retentional signals, need to go back to the ER.

    • Proteins tagged with M6P are sent to the lysosomes

      
      

Proteins that need to be secreted from the cell:

• have distinct signal sequences that are specific for the cargo in the vesicle

• These proteins can be produced by the cell and are needed for proper function of the body (ex. hormones)

STEPS OF TRANSLOCATION INTO THE ER

1. Signal Sequence:

2. SRP Binding

3. Ribosome Docking

4. Translocation

TRANSLOCATION INTO THE ER : Signal Sequence

the presence of a signal that is translated as part of the protein, termed the ER signal sequence.

TRANSLOCATION INTO THE ER : signal recognition particle (SRP)

• This sequence, as it emerges from the ribosome, interacts with a receptor, termed the signal recognition particle (SRP), which binds to the ribosome that is translating the protein. This pauses translation. The binding of GTP simply indicates this is an energy consuming process.

TRANSLOCATION INTO THE ER :Ribosome Docking

During the pause in translation, the ribosome docks onto the ER membrane by the SRP interacting with an SRP receptor and a complex called the translocon.  This allows and facilitates the translocation of the growing protein into the ER.

TRANSLOCATION INTO THE ER :Translocation

Translation restarts, and once the protein has translocated into the ER, the signal peptide sequence is cleaved off the protein, translation finishes, and the protein folds inside the ER lumen. The finished protein is destined to be soluble and not attached to a membrane.

Translocating proteins destine to be EMBEDDED INTO THE LIPID MEMBRANE

• Transmembrane Domain:  the transmembrane signal anchor sequences initiates the process for the protein to become embedded in the lipid bilayer.

• Protein Enters Lipid Bilayer: The transmembrane signal sequence becomes embedded in the lipid bilayer and will be the transmembrane domain of the protein once it is a functional mature protein within a membrane.

POST TRANSLATIONAL MODIFICATIONS OF PROTEIN BY THE ER 

The modifications may include:

• The addition of proteins, sugars, lipids, and new functional groups like phosphates and methyl groups, which can change the final target location, structure, or function of the protein.

• The cleaving (cutting) of the peptide bonds in the protein.

• The ER signal sequence, typically located at the N-terminus of a protein, is removed

ENZYMES INVOLVED IN PROTEIN FOLDING

• Protein Disulfide Isomerase (PDIs)

• Binding Protein

Binding Protein

• Another class of protein, termed chaperonins, help fold the polypeptide by binding hydrophobic patches in recently translated proteins

• One type of chaperonin is called BiP. BiP brings these patches together to help fold them correctly into the hydrophobic interior of mature proteins. Once buried in the protein folds, BiP can no longer access the hydrophobic patches.

Protein Disulfide Isomerase (PDIs)

• Help form disulfide bonds between the thiol groups in the side chains of cysteines (an amino acid). These disulfide bonds help the protein to fold properly and stabilize it.

PEPTIDE BOND

The carboxyl group of one amino acid reacts with the amino group of the other amino acid.

This is a dehydration reaction. One molecule of water is released from this reaction

Ends of Amino Acid Chains

• The one with an amino group free, called the amino terminus (N-terminus)

• The one with a carboxylic acid group free, called the carboxy terminus (C-terminus)

Primary Protein Structure

• The linear amino acid/protein sequence

• Amino acid numbering starts at the amino terminal end and finishes at the carboxy terminus (N to C)

Secondary Protein Structure

• A region of organization in the peptide sequence 

• Examples of common secondary structures:

  • Alpha Helix: Hydrogen bonds between every fourth amino acid causes a coiled appearance

  • Beta Sheets: Hydrogen bonds between the backbones of amino acids causing planes to be formed. They can be parallel (N to C for both rows) or anti-parallel (direction of peptides switch between rows)

Tertiary Protein Structure

• Molecular chaperones are needed to help facilitate protein folding, ensuring the protein gets the right shape and are not misfolded

• Disulfide bonds form in the tertiary structure

Quaternary Protein Structure

Multiple proteins are assembled into a complex. The individual proteins are called subunits of a quaternary structure only if they cannot have a function outside the complex.

DOMAINS

a building block of a protein structure. Some protein domains have clear and specific functions, these domains will often keep doing their jobs even when inserted into a protein.

• A discrete structural unit that is assumed to fold independently from the protein and therefore has its own function

• Can be made from 20-100s of amino acids, are made from many secondary structures

• Most proteins are multi-domain, and domains are often conserved between evolutionarily related proteins.

Protein changes: covalent modifications

• Relatively long lasting

• Adding phosphate groups, methyl groups, or acetyl groups are all methods of changing protein shape. These will activate or inactivate proteins, or change how they can interact with other proteins in the cell

• Disulfide bonds and the addition of lipids or sugar structures are example

Protein changes: non-covalent modifications

• Relatively short lived

• Can include proteins interacting with each other in binding sites or small molecules like oxygen, calcium or magnesium binding transiently

Question 1: Compare and contrast the levels of DNA packaging to protein folding.

Although both proteins and DNA have a similar number of levels, the end goals are different. DNA needs to be carefully stored, organized, and read safely in the nucleus when it is needed. In contrast, proteins need to be folded to be able to do a large variety of different tasks in the cell, and may be sent outside the cell depending on the function of the proteins.

Why do you think the cell does not allow cargo to free float between organelles?

Without vesicles, organelles would be isolated from one another and would not have the ability to shuttle cargo between them. It would also leave the cell unable to bring in cargo from the extracellular space or secrete cargo out of the cell.

What structure is responsible for anchoring the ribosome to the surface of the RER and reinitiating translocation?

The translocon facilitates the docking of a ribosome to the surface of the RER and reinitiates translocation into the lumen of the ER.

STEPS OF VESICLE TRAFFICKING IN THE ENDOMEMBRANE SYSTEM 

1. 1a. Cargo Selection 

2. 1b. Coat Proteins 

3. Budding 

4. Scission

5. Uncoating

6. Transport

7. Tethering

8. Docking

9. Fusion

10. Disassembly

1a. Cargo Selection 

• Cargo, meaning proteins, lipids or other macromolecules, is gathered in the area of the membrane where the vesicle will be made

  • Signal sequences and receptors help to ensure the correct cargo comes to the location and efficiently

1b. Coat Proteins

• Coat proteins from the cytosol bind to the area of the membrane that will become the outside of the vesicle. This causes receptors and other proteins to cluster on the surface of the membrane.

• There are three common coat proteins:

  • COPII

  • COPI

  • Clathrin

Budding

Coat proteins interact with cytosolic adaptor proteins, the proteins work to pull the membrane creating a bulge shape that will become the vesicle

Scission

The protein Dynamin helps the vesicle to be pinched off from the membrane and to be released into the cytoplasm

Uncoating

Now that the vesicle has successfully been made, the coat proteins are disassembled and can be reused

Transport

• Most vesicles are attached to a motor protein that allows for movement within the cell

• The vesicle will move along the microtubules of the cytoskeleton, allowing the cell to control where the vesicle goes

Tethering

A tether protein is used to attach to a receptor on the acceptor membrane, and bringing the vesicle closer to the acceptor target proteins

Docking

The vesicle docks on the surface of the acceptor target protein

Fusion

The vesicle membrane becomes continuous with the acceptor membrane. This occurs due to pairs of v-SNAREs and t-SNAREs interacting. This allows for the contents of the vesicle to be expelled into the acceptor organelle.

Disassembly

The tether and fusion proteins and receptors disassemble and go back to their original organelle