Cell Molec Unit 2

Factors that determine the rate of diffusion

Charge/polarity and size. In this order:

1. Hydrophobic molecules

2. Small uncharged polar

3. Large uncharged polar

4. Ions

Transport proteins types

Transporters: possess a solute-binding site and have a conformational change in the transporter after the solute binds

Channels: interact with solute more weakly, allow solutes to move downhill with gradients, do not bind to solutes

Define Passive Transport, list examples of simple diffusion.  What is facilitated diffusion?  List example

Passive transport: Diffusion of a molecule down its concentration gradient requiring no energy usage

Simple diffusion examples:

Oxygen in lungs

CO2 from body

Facilitated diffusion: passive transport with the help of channel proteins or transporters without the usage of ATP

Examples:

GLUT

Active transport and the difference between primary and secondary transport.

Active transport: moves molecules or ions against their gradient

Primary active transport: uses ATP hydrolysis to move molecules

Example:

• Complete the chart from class about transporters

  • Under review folder

• Primary active is all the ATP driven pumps

  • P, ABC, and v-type

  • Only pump cycle in detail is the calcium pump

Secondary active transport: uses an existing concentration gradient made by a primary active transport; uses symporters and antiporters

Example: Na and Glucose pump

ATP driven pumps and structures

P-type pumps - self-phosphorylation during the cycle, used for Na+, H+, and Ca2+ pumps

ABC transporters - transport nutrients, toxins, etc

V-type pumps - H+ to acidify

Structures:

• P-type pumps structure allows for a site to phosphorylate; only one ATP binds

• ATP have two binding sites instead of one, opens like scissors

• V type know theres a domain for ATP to bind

Describe Na⁺K⁺ Pump (P type)

Concentration of K+ is higher outside the cell than inside, and reverse for Na+. This pump is very important for the Na+ gradient drives transport of nutrients. ⅓ of cells energy devoted to this pump.

ABC transporters (list examples).  Role of ATP binding and hydrolysis in transporter conformational changes

Examples: Bacterial ABC transporter, Eukaryotic ABC transporter

Eukaryotic examples: P-glycoprotein in cancer cells makes them resistant to chemo; Plasmodium falciparum that causes malaria pumps out chloroquine

DNP as an uncoupler

• Collapse of the proton gradient on both sides

• Increases oxygen consumption, metabolic rate, liver failure and hyperthermia

Aquaporin, how conventional ions are blocked and how H+ ions are blocked.

How conventional ions are blocked: the narrowest point of the pore, the arginine-selective filter, has steric boundaries for some ions.  Ions are surrounded by hydration shells as well.

In water, the H+ ions move down a chain by making a series of H bonds. Aquaporins force single-file water molecules to pass through in a strict, specific orientation dictated by conserved Asparagine-Proline-Alanine (NPA) motifs and local dipoles. This orientation breaks the continuous hydrogen-bond wire, stopping proton conduction

Ion channels vs pores

1. Ion channels are selective for ions

2. Ion channels are not continuously open

Action Potential Na Conformational States

Open:  Na+ channels are open when the membrane is depolarized in response to a stimulus. From here, a small influx of charge depolarizes it further, causing a rush of Na+ in

Inactivation: The channel pore is quickly blocked by an inactivation particle (often described as a hinged lid or inactivation gate) shortly after opening, preventing any further sodium ion flow despite the continued depolarization of the membrane. This phase halts the inward sodium current, ends the peak of the action potential, and establishes the absolute refractory period.

 

Closed: The activation gate is shut while the inactivation gate is open, resting the channel in a non-conducting state at the baseline negative membrane potential. From here, the channel is ready to respond to a new depolarizing stimulus, dictating the relative refractory period and the resting phase of the cell.

Action Potential

1. Inside a neuron is more negative than the outside of the cell.

2. Na+ channels are open when the membrane is depolarized in response to a stimulus.

3. This influx of Na+ ions makes the gradient more positive. The channel pore becomes blocked by the inactivation particle

4. K+ channels open to move K+ out of the cell to restore membrane potential. Overshoot and cause repolarization.

What is CFTR gene

• Glycoprotein found in cell membranes

• ABC transporter

• Pumps Cl- ions out of cell

What areas of the body are most affected by CFTR gene

CFTR is found in epithelial cells, which cover most of the body's internal and external surfaces. CFTR is responsible for the release of chloride from the cell. When chloride cannot leave the cell, water goes in due to the increased ion concentration. This makes the mucus on the surface of the lungs and pancreas thicker. 

In the lungs, the cilia cannot move freely and remove debris since the mucus is so thick, causing obstructions. In the pancreas, the mucus blocks the digestive enzymes from reaching the small intestine. This causes malabsorption and pancreatic scarring.

How is CF diagnosed

1. Meconium ileus at birth

2. Blood test for pancreatic enzyme levels

3. Sweat test

4. Genetic test

Treatment for CF

1. Antibiotics

2. Loosen mucus with vest

3. Medications with digestive enzymes

4. Lunch transplant

5. Targeted gene therapy

6. Kalydeco

   1. For a certain mutation where it is in the right place in the cell but is locked

7. Trikafta

   1. Helps channels stay open longer for a certain mutation affecting 90% of people

8. Gene therapy

   1. Trial in the UK

3 Mechanisms for sorting proteins

Gated transport - nuclear pore complexes

Vesicle transport - cytosol to mitochondria, cytosol to peroxisomes

Transmembrane transport - all else

Two types of sorting signals

• Signal peptides 15-60 aa that direct proteins

  • ER, mitochondria, peroxisomes, chloroplasts

• Signal patches 3D arrangement of aa into a patch

  • Golgi, lysosome

Movement through a nuclear pore complex

1. Importin receptor binds to cargo protein

2. Importins then bind to FG in the unstructured domain channel and “walk” down

3. Binding of the cargo and receptor to Ran-GTP releases the protein

4. Important and Ran GTP move up through the pore again

5. Ran GTP is hydrolyzed into Ran GDP and dissociates

6. Importin is empty

Origin of endosymbiosis theory

1. Both container their own genomes of circular DNA 

2. Mitochondria replicate with binary fision

3. Mitochondria inner membrane resembles that of plasma membrane

4. Mitochondria have ribosomes

Translocators of the mitochondria and their main functions

TOM - outer membrane. Imports all the mitochondrial proteins

SAM - outer membrane. Help fold beta barrels

TIM 22- inner membrane, mediates insertions of a subclass of IM proteins

TIM 23 - inner membrane, helps insert transmembrane proteins and transports some proteins into the transmembrane space

OXA - insertion of inner membrane mitochondria synthesized proteins

Three specific areas of protein import into mitochondria that require input of energy

1. Removal of HSP from protein in the extracellular space

2. Maintaining the electrochemical gradient

3. Removal of HSP in mitochondria

Smooth ER, Rough ER, transitional zone

Smooth ER: site of vesicle budding and transitional zones

Transitional zones: where newly synthesized proteins are packaged to leave the ER

Rough ER: primary site of protein synthesis of transmembrane proteins and soluble secreted proteins.

Blobel Experiment

Blobel wanted to figure out if there was a zip code signal attached to proteins to direct them where they needed to go. To do this, he added microsomes (small pieces of ER) to one tube and one without. When the mRNA, ribosomes, and amino acids were added, the tube without the microsomes synthesized proteins larger than those in the tube with microsomes. This is because the signal is trimmed off once embedded in the microsomes.

SRPs

SRP is the signal recognition particle, and it binds to the signal sequence and then to a receptor on the ER surface. The signal sequence is has 8 or more non-polar amino acids at its center.

Two populations of ribosomes

Free ribosomes are throughout the cytosol. They synthesize proteins intended to be used inside the cell. There are also membrane bound ribosomes, that exist on the ER membrane. These make proteins intended to be exported.

Sec61

A heterotrimer that forms a gated channel at the core of a translocator. In the ER membrane, the “plug” seals the channel from the inside when no protein is being transported. When a ribosome docks on the ER membrane and begins translating a protein with an appropriate signal peptide, the signal sequence wedges into the seam, opening it.

translocation of soluble proteins in ER

1. ER binds to a specific part of the pore, which is a start transfer signal

2. The signal sequence remains in contact with the pore during translocation

3. Signal sequence also in contact with the seam of Sec61

4. Peptidase cleaves signal, protein released into the lumen

Glycosylation

• Oligosaccharyl transferase mediates the transfer of an oligosaccharide to the Asn residue of a protein

• The enzyme is closely associated with SEC61

• Dolichol is embedded in the ER membrane to build the precursor of the oligosaccharide on

Retrotranslocation mechanism

In the endoplasmic reticulum (ER), mannose trimming acts as a structural "timer" that marks terminally misfolded glycoproteins for retrotranslocation (dislocation) into the cytosol.

3 pathways of vesicular transport

1. secretory pathway - out from ER and Golgi

2. endocytic pathway - inward from the membrane

3. retrieval - balances the flow of membranes

Structure and function of the Golgi apparatus

Golgi is made up of cisternae, which are short pancake like stacks

Golis purpose os to transport modify and package proteins and lipids

Coated vesicles and their functions

1. Clathrin coated

   1. From plasma membrane to endosomes, or from Golgi to endosomes

   2. Inner coat of adaptor proteins that attach clathrin directly to the cargo

2. COP1

   1. Transport from Golgi to ER, intragolgi

3. COP2

   1. ER to Golgi

Process of Membrane Fusion

Rab proteins: 

• Transport vesicles display specific surface markers to ensure they fuse only with the correct target membrane. Rab are GTPases

• Rab GDP when protein soluble, and Rab GTP when bound to a membrane

• The active Rab-GTP on an incoming transport vesicle binds to a specific Rab effector located on the target membrane
  
  

SNARE:

• Once tethered, docking is mediated by specific transmembrane proteins called SNARE

• The interaction between v- and t-SNAREs (vesicle and transfer) forms a stable, tight four-helix bundle known as a trans-SNARE complex

• These helicies wrapped tightly around each other squeeze water out 

• Pried apart before another round

Homotypic Fusion

Homotypic fusion is the fusion of two membranes derived from the same compartment; it happens when transport vesicles in the ER fuse.

KDEL and affinities

Soluble ER proteins contain a KDEL sequence; the KDEL receptor moves between the Golgi and ER to return ER proteins back. KDEL receptor has high affinity in the acidic Golgi environment, and low affinity in the ER, since the escape proteins are already returned home and can be released.

Why are proteins glycosylated in golgi?

Cisternal maturation and vesicle transport model

• Cisternal Maturation Model: The Golgi cisternae are dynamic structures. New cis-cisternae form from fused ER-derived vesicles. These cisternae physically progress and mature from the cis face to the trans face, changing their enzyme composition over time while carrying forward cargo inside.

• Vesicular Transport Model: The Golgi cisternae are stationary, permanent compartments. Cargo proteins move forward (anterograde) from one cisterna to the next via budding and fusing transport vesicles, while the structural layers of the Golgi stay in place

Why are membrane proteins glycosylated?

Lysosome maturation

Lysosomes are heterogeneous because they differ in size, shape, and contents.

Endolysosomes are formed when late endosomes combine with lysosomes for digestion. It matures into lysosomes once hydrolases complete digestion.

Four pathways for degradation in lysosomes

1. Endocytosis - from the membrane

2. phagocytosis - bacterium from surface

3. macropinocytosis - solutes from surface

4. autophagy - cytosolic compartments

Autophagy

Autophagy - eating itself, helps restructure differentiating cells

Digests large objects proteosomes can’t handle

Selective - targeted for cell components that may be damaged or harmful

Nonselective - begins digesting any cell component as a response to starvation, happens in newborns

Segregation of hydrolases in the trans golgi network, and how M6P works.

1. M6P attached

2. M6P binds to the receptor in the trans golgi network

3. Protein is put into a clathrin-coated vesicle

4. Transported to the endosome

5. In the early endosome, the acidic environment dissociates M6P receptors

6. These return back to the Golgi

I-Cell Disease

Lack of hydrolytic enzymes in many types of cells. Causes a buildup of waste.

Endosome Maturation

1. Vesicles form near plasma membrane

2. They combine to form early endosomes

3. Internal cargo is sorted

4. Some return to plasma membrane

5. Once recycling is over, they lose the tubular domain and become more acidic, and hydrolases are active

Endocytosis of LDL

1. Receptor-mediated endocytosis

2. Uncoated from clathrin

3. Fusion with early endosome

4. Receptor is recycled

5. Late endosome

6. Endolysosome digests the LDL, releases cholesterol

Protein sorting in trans golgi network

1. Signal-mediated diversion to lysosomes with endosomes

2. Signal-mediated diversion to secretory vesicles for regulated secretion

3. Constitutive secretory pathway

Regulated secretion

secretory vesicles release contents when a singal is recieved, like a hormone or nerve impulse.

synaptic vesicles

specialized secretory vesicle, stores nuerotransmitters.

Exocytosis of a synaptic vesicle

Docking: The synaptic vesicle docks at the presynaptic plasma membrane, bringing the v-SNARE (synaptobrevin) close to the t-SNAREs (syntaxin and SNAP-25).

Priming I: The SNAREs partially pair up to form a partially assembled SNARE bundle, though the helices are not yet fully wound.

Priming II (Complexin-Blocked): Regulatory proteins called complexins bind to the SNARE bundle and "freeze" it, preventing premature membrane fusion.

Fusion Pore Opening: An action potential arrives at the nerve terminal, triggering an influx of calcium ions (Ca2+) through voltage-gated Ca2+ channels. The rise in calcium causes synaptotagmin (via its Ca2+-binding domains) to bind to phospholipids and SNAREs, displacing the complexins and opening the fusion pore.

Fusion Complete: Membrane fusion is completed, and neurotransmitters are released into the synaptic cleft.