T3 L9: Protein and vesicular transport

What does mislocalisation of proteins contribute to?

Mislocalisation of proteins contributes to disease (e.g. lysosomal storage disorders).

To stay organised, cells use two strategies:

1. Large protein complexes (biochemical sub-compartments).

2. Membrane-bound organelles (e.g. nucleus, ER, mitochondria).

Learn this

What does a selectively permeable membrane do?

Membranes which form a selectively permeable membrane to control the entry and exit of most molecules

Label a cell of membrane enclosed organelles

How are organelles separated?

Organelles can be separated by differential centrifugation, allowing their proteins and functions to be studied in isolation.

What can an isolated mitochondria still do?

For example, isolated mitochondria can still generate ATP from pyruvate when supplied with ADP, phosphate, and oxygen.

Functions of the nucleus

• Contains genetic material (DNA);

• site of transcription

• enclosed by double membrane (nuclear envelope)

• with pores (nuclear pores) for communication with cytosol

Function of ER

Continuous with the outer nuclear membrane; the major site of lipid and protein synthesis

Functions of rough ER

• Studded with ribosomes;

• site of synthesis of proteins destined for membranes, secretion, or organelles;

• proteins inserted into ER lumen or membrane

Functions of smooth ER

• Lacks ribosomes;

• synthesises lipids (e.g. steroid hormones);

• detoxifies organic molecules (e.g. drugs, alcohol);

• sequesters and releases Ca²⁺ (important in muscle contraction)

Functions of golgi apparatus

• Receives proteins and lipids from the ER;

• modifies, sorts and packages them for either secretion or delivery to another organelle

Functions of lysosomes

• Small sacs of digestive enzymes which degrade worn-out organelles,

• as well as macromolecules and particles taken into the cell by endocytosis.

Functions of peroxisomes

• Small organelles that contain enzymes that break down lipids and destroy toxic molecules, producing hydrogen peroxide

Functions of endosomes

Sort the ingested molecules and recycle some of them back to the plasma membrane.

Functions of mitochondria

Site of ATP synthesis and oxidative phosphorylation

Which 3 mechanisms are proteins transported into organelles?

1. Through nuclear pores

2. Across membranes

3. By transport vesicles

Describe through nuclear pores

Proteins move from cytosol into the nucleus via nuclear pores spanning both nuclear membranes

Describe across membranes

Proteins cross the ER, mitochondria, or peroxisome membranes using protein translocators

Describe by transport vesicles

Proteins leaving the ER and moving within the endomembrane system are carried in transport vesicles

Describe the proteins during transport

• Transport of proteins requires energy

• Proteins transported through pores or by vesicles normally remain folded

• Proteins transported across or into membranes must first be unfolded

What do signal sequences do?

• Signal sequences direct proteins to the correct compartment

• The typical sorting signal on a protein is a continuous stretch of amino acid sequence, typically 15–60 amino acids long

• Signal sequence is often (but not always) removed from the finished protein once it has been sorted

• Signal sequences can vary despite specifying the same destination (physical properties like hydrophobicity more important)

What occurs when proteins do not have a signal sequence?

Remain in the cytosol and dont go to appropriate organelle

Learn the table - normal signal sequence vs relocated signal sequence

Synthesis of all proteins occurs on

ribosomes

Steps of protein sorting in the ribosmes

1. Ribosomal subunits form a common pool in the cytosol

2. Ribosomes start free in the cytosol. Some stay free; others are directed to the ER if the protein has an ER signal sequence 

ER signal sequence → ER targeting

No signal sequence → remain in cytosol

3. Mitochondria have their own ribosomes but most of their proteins are made in the cytosol

4. Proteins synthesized by ribosomes in the cytosol either remain in the cytosol or get transported into mitochondria, peroxisomes or the nucleus.

5. Ribosomes at the Endoplasmic Reticulum produce proteins destined for secretion, the plasma membrane, secretory vesicles and lysosomes.

6. Proteins made at ER are sorted in the Golgi apparatus

Learn diagram for protein sorting

Learn diagram for protein sorting - free ribosomes in cytosol

Learning diagram for protein sorting - membrane-bound ribosomes

What are the exceptions for protein synthesis and sorting?

The exceptions are the few mitochondrial and chloroplast proteins that are synthesized on ribosomes inside these organelles

Summary of protein sorting

• Every protein begins synthesis on cytosolic ribosomes

• Its signal sequence acts like a postcode, directing it to the correct organelle/location

• Free ribosomes make proteins that stay in the cytosol or are imported into the nucleus, mitochondria, or peroxisomes

• ER-bound ribosomes make proteins for secretion, membranes, or the endomembrane system

Transport into the nucleus

• The nuclear envelope is formed by two concentric membranes (inner and outer nuclear membrane)

• The nuclear membrane is perforated by nuclear pores

• Only small molecules can diffuse into the nucleus

• Proteins such as transcription factors are synthesized at the cytoplasm and need to be transported into the nucleus

• Proteins require specific signals to actively transported across nuclear pores

• Proteins are translocated in their fully folded conformation

What are nuclear pores?

Nuclear pores are a complex of around 30 different proteins termed nucleoporins

What are the functions of nuclear pores?

• Nuclear pores pass through both nuclear membranes controlling what can pass into and out of the nucleus

• The center of the pore is lined with protein which are largely unstructured forming a “kelp-forest” like barrier to larger molecules but allowing small molecules to diffuse through

Steps of nuclear import

1. Newly made proteins destined for the nucleus need to have an appropriate sorting signal called a nuclear localisation signal

2. The localisation signal is recognised by a nuclear import receptor called Importin that interacts with the cytosolic fibrils that extend from the rim of the pore

3. After being captured, the receptors with their cargo jostle their way through the gel-like meshwork formed from the unstructured regions of the nuclear pore proteins

4. Binding of Ran-GTP to Importin triggers the release of the cargo in the nucleus and importin returns to the cytosolic side

5. Nuclear export receptors (exportins) recognise nuclear export signals and work in a similar way, driving protein and RNA traffic from the nucleus to the cytosol

Steps of mitochondrial transport

1. Mitochondria also have two membranes. Signal sequence is recognised by an import receptor on outer membrane

2. Import receptor is associated with a protein translocator

3. Import receptor and protein translocator diffuse to an area where membranes come into close proximity to engage second translocator

4. Proteins translocator passes the protein through the membranes

5. Each protein is unfolded during transport, then refolds once inside, where its signal sequence is removed after translocation

Summarise nuclear transport and mitochondrial import

Nuclear Transport:

Proteins enter the nucleus folded via nuclear pores

Require a nuclear localisation signal (NLS) recognised by importins

Ran-GTP mediates cargo release and receptor recycling

Mitochondrial Import:

Proteins are transported unfolded across both membranes via translocators

Signal sequence is cleaved and protein refolds inside

Which organelle performs protein sorting?

Endoplasmic reticulum

How does signal recognition particle directs ribosomes to ER

1. Proteins with an ER signal sequence are recognised during translation

2. SRP (signal recognition particle) binds the signal and ribosome, pausing translation

3. The complex docks at the SRP receptor on the ER membrane

4. Ribosome is transferred to a protein translocator channel (forming regions of rough ER).

5. Translation resumes, and the growing protein is threaded into the ER lumen or membrane

Steps of protein import into the ER lumen

1. Protein translocator binds the signal sequence during translation and threads the polypeptide across the lipid bilayer as a loop

2. The signal peptide is removed from the growing polypeptide by signal peptidase and left in the membrane to be degraded

3. As the polypeptide crosses the membrane the molecular chaperone BiP binds ready to help fold the protein within the ER

4. Once protein synthesis is complete, the polypeptide is released as a soluble protein in the ER lumen and the protein translocator closes

Do all ER-bound proteins enter the lumen?

Not all ER-bound proteins enter the ER lumen, some become anchored in the ER membrane (transmembrane proteins)

Steps of insertion of proteins in the single pass transmembrane protein?

1. The N-terminal ER signal sequence (red) directs the ribosome to the ER and initiates the transfer of the polypeptide chain through the ER membrane

2. A stop-transfer sequence (orange) within the growing chain pauses movement through the translocator

3. The N-terminal signal sequence is removed by a signal peptidase and the protein translocator releases the growing peptide laterally into the membrane

4. The hydrophobic stop-transfer sequence remains in the bilayer as an α-helical segment, anchoring the protein in place

Steps of insertion of proteins in the double pass transmembrane protein?

1. Some proteins use an internal signal sequence (red) that acts as both a start-transfer signal and an anchor in the membrane

2. Internal signal sequence is recognised by SRP, which brings the ribosome to the ER membrane

3. When a stop-transfer sequence (orange) enters the translocator, both start- and stop-transfer sequences are released sideways into the lipid bilayer

4. Neither sequence is cleaved off, so the protein remains embedded as a double-pass transmembrane protein

Steps of protein modification via glycosylation

1. During transport into the ER lumen or ER membrane many proteins are glycosylated

2. When an appropriate asparagine (Asn) enters the ER lumen, it is glycosylated by the covalent addition of a branched oligosaccharide side chain

3. Glycosylation is critical for physiological and pathological cellular functions of many proteins

4. Changes in glycosylation can modulate inflammatory responses, enable viral immune escape, promote cancer cell metastasis or regulate apoptosis.

5. Very few proteins in the cytosol are glycosylated, and those that are have only a single sugar attached to them.

Which proteins in the cytosol are glycosylated?

Very few proteins in the cytosol are glycosylated, and those that are have only a single sugar attached to them.

Steps of unfolded protein response (UPR)

1. Misfolded proteins are actively retained in the ER by binding to chaperone proteins

2. Chaperones prevent misfolded proteins from aggregating and helps steer proteins along a path towards protein folding

3. An excess of unfolded proteins in the ER triggers the unfolded protein response (UPR)

4. Sensor proteins become activated by the accumulation of unfolded proteins and then stimulate an expansion of the ER, increase number of chaperones and reduce the amount of new proteins entering the ER.

Summarise of ER import, glycosylation, UPR

ER Import:

Signal recognition particle (SRP) pauses translation and directs the ribosome to the ER

Proteins are threaded into the ER lumen or inserted into the membrane during translation

Glycosylation:

Addition of branched oligosaccharide chains to asparagine (Asn) residues in the ER

Critical for proper folding, stability, immune recognition, and cell signalling

Unfolded Protein Response (UPR):

Triggered by accumulation of misfolded proteins in the ER

Increases chaperone expression, expands the ER, and reduces new protein synthesis to restore homeostasis

Steps of vesicular transport

1. Vesicular transport allows materials to exit or enter the cell

2. Extends outward from the ER to the plasma membrane

3. Exocytosis: vesicles fuse with the plasma membrane releasing their contents outside the cell

4. Reaches inward from the plasma membrane to lysosomes

5. Endocytosis: vesicles bud inwards from the plasma membrane and are carried into the cell

2 pathways for vesicle budding and membrane fusion

1. Secretory pathway

2. Endocytic pathway

Summarise secretory pathway

• Major secretory pathway starts with synthesis of proteins on the ER membrane and their entry into the ER, and leads through the Golgi to the cell surface

• A side branch leads through endosomes to lysosomes

Summarise endocytic pathway

Vesicles derived from the plasma membrane are delivered to early endosomes and usually on to lysosomes via late endosomes

Two pathways of secretory pathway

1. Constitutive pathway

2. Regulated pathway

Describe constitutive pathway

Continuous secretion of soluble proteins from the cell. Supplies plasma membrane with newly made lipids and proteins.

Describe reguated pathway

Operates only in cells that are specialised for secretion. Secretory vesicles store product until an extracellular signal that stimulates them to fuse with the plasma membrane and release their contents by exocytosis.

What are coat proteins and why is it advantageous?

Vesicles that bud from membranes usually have a distinctive protein coat on their cytosolic surface (coated vesicles)

• Helps shape the membrane into a bud

• Directly or indirectly capture molecules for onward transport

What occurs after budding?

After budding the coat is shed, allowing its membrane to interact directly with the membrane to which it will fuse

Types of coated vesicles

Steps of Clathrin coated vesicle formation

1. Clathrin-coated vesicles bud from the Golgi apparatus on the outward secretory pathway and from the plasma membrane on the inward endocytic pathway

2. Clathrin molecules are made up of three chains which assemble into a basketlike cage on the cytosolic surface of the vesicle membrane

3. At the plasma membrane, each vesicle starts off as a clathrin-coated pit which are concentrated with receptors

4. Clathrin is only involved in shaping the budding membrane, other proteins called adaptins select the cargo molecules

Steps of Clathrin-coated vesicles transport selected cargo molecules

1. Cargo receptors, with their bound cargo, are captured by adaptins

2. Adaptins also bind clathrin molecules to the cytosolic surface of the budding vesicle

3. Clathrin starts shaping the membrane into a vesicle

4. Dynamin (a GTP binding protein) assembles around the neck of budding vesicles to pinch off the vesicle

5. After budding the coat proteins are removed and the naked vesicle can fuse with its target membrane

Steps of vesicles at target membrane 

1. Once a vesicle has reached its target, it must recognise and dock with its specific organelle

2. This identification process is dependent on a family of proteins called Rab proteins

3. Specific Rab proteins on the surface of each vesicle are recognised by corresponding tethering proteins on the surface of the target membrane

4. Once the tethering protein has captured a vesicle by binding to its Rab protein, SNAREs on the vesicle (v-SNAREs) interact with complementary SNAREs on the target membrane (t-SNARES) and firmly dock the vesicle

5. After a transport vesicle buds from a membrane, it must find its way to the correct destination to deliver its contents. Often, the vesicle is actively transported by motor proteins that move along cytoskeletal fibers

Steps of membrane fusion

1. The same v-SNAREs and t-SNAREs bring the two lipid bilayers into close opposition

2. The force of the SNAREs winding together squeezes out any water molecules that remains trapped between the membranes (needs to occur for membranes to come within 1.5nm of each other!)

3. This close contact allows the lipids of the two membranes to flow together to form a continuous bilayer (other proteins help complete this fusion process)

4. After fusion SNAREs are pried apart so they can be used again

Summary of vesicular transport

Moves materials between organelles and to/from the plasma membrane

Exocytosis: delivers cargo to the cell surface or extracellular space

Endocytosis: internalises material from the plasma membrane

Summary of secretory pathways

Constitutive: continuous delivery of proteins and lipids to the plasma membrane

Regulated: secretion triggered by external signals via stored vesicles

Summary of coated vesicles

Clathrin shapes the budding membrane

Adaptins select cargo by binding to cargo receptors

Dynamin (GTPase) pinches off vesicles from the membrane

After budding, the coat is shed to allow fusion

Summary of vesicle targetting and fusion

Rab proteins on vesicles bind tethering proteins on target membranes

SNAREs (v-SNAREs and t-SNAREs) ensure specific docking and drive membrane fusion

Function of phagocytosis

Most extreme type of endocytosis is known as phagocytosis

Phagocytic cells such as neutrophils and macrophages defend us against infection by ingesting invading microorganisms

Phagocytic cells extend pseudopods (sheet-like projections of the plasma membrane) to engulf their target

Form a phagosome which then fuses with a lysosome, where the target is destroyed

Viruses can enter cells via receptor-mediated endocytosis - Describe

• Viruses taken up by receptor-mediated endocytosis will fuse with lysosomes, where the low pH releases the viral genome into the cytoplasm

• Receptor-mediated endocytosis can also be exploited by viruses

  E.g. Influenza virus , HIV virus and SARS-CoV-2 virus enter the cells this way

Receptor translocation

Degradation

Receptors are delivered to lysosomes to be degraded

Recycling

Receptors are returned either to the same plasma membrane domain from which they come

Transcytosis

Receptors are returned to a different domain of the plasma membrane

Lysosomes are the principal sites of intracellular digestion - Explain

Lysosomes are membranous sacs of hydrolytic enzymes that carry out controlled intracellular digestion

Materials destined for degradation in lysosomes follow different pathways

Phagocytosis: where specialised cells take up large particles

Endocytosis: the uptake of material from outside the cell often induced by receptor binding

Autophagy: the breakdown of the cells own proteins and organelles by enclosing them in a membrane that fuses with the lysosome