Cell sorting Endocytosis Exocytosis

Question 1

where are proteins located?

Answer 1

Specialised function
requires proteins:

 Eukaryotes – proteins play a crucial role in various cellular functions across multiple sub-
cellular compartments. e.g. structural, enzymatic function, gene expression, cell communication, etc.

 Nucleus needs nuclear
proteins- they serve as the repository (storage) for genetic material (DNA) and play a role in ensuring the proper functioning and regulation of genetic material e.g. they are involved in processes such as transcription(RNA processing), DNA replication, DNA repair, gene regulation and cell cycle regulation.

 ER needs ER proteins- they ensure proper functioning protein folding, protein modification, synthesis of membrane and secretory proteins, lipid synthesis, calcium storage and detoxification of harmful compounds.

Question 2

once proteins are synthesised in ribosomes, they must be directed to their proper location e.g. nucleus or Golgi apparatus. How do proteins know where to go?

Answer 2

Proteins are guided primarily by their amino acid sequences known as sorting signals or address sequences (These sequences act like postal codes, ensuring that proteins are delivered to the right place).

Sorting Signals:
1- Certain proteins are synthesized with short stretches of amino acids that function as sorting signals.
2- These signals are recognized by cellular machinery (such as receptor proteins or translocons- protein complexes that facilitate the transport of proteins across cellular membranes, particularly in the endoplasmic reticulum (ER) and other organelles. ) that help direct the protein to its intended destination.
3- Examples include mitochondrial targeting signals, nuclear localization signals, and endoplasmic reticulum signal sequences.

Question 3

Why do some proteins have no sorting signals and what happens to them.

Answer 3

Cytosolic Proteins:
A significant portion of proteins synthesized on free ribosomes in the cytosol do not have sorting signals, meaning they remain in the cytosol and function there- These proteins often play roles in metabolic pathways, signal transduction, and other essential cellular processes within the cytosolic environment.

Question 4

Other than sorting signals, what else can proteins have.

Answer 4

Some proteins have signal sequences- short peptide (amino acid) sequences at the N-terminus of proteins- they facilitate the localization (reach the right place) and proper functioning of proteins by directing them to their intended destination within or outside the cell, such as the (ER), mitochondria, or extracellular space.

Question 5

Types of signal sequences.

Answer 5

Signal Peptides: Direct proteins to the ER for secretion or for incorporation into membranes.

Mitochondrial Targeting Sequences: Direct proteins to the mitochondria.

Nuclear Localization Signals: Facilitate the transport of proteins into the nucleus.

Question 6

what happens to the localisation of t antigens when the address is changed?

Answer 6

The protein may not be correctly targeted to its intended destination. This can lead to accumulation of the protein in the cytoplasm or in an incorrect organelle.

Question 7

what are the three main stages of protein traffic (the processes and mechanisms by which proteins are synthesized, modified, sorted, and transported within a cell).

Answer 7

1- transport through nuclear pores
2- transport across membranes
3- transport by vesicles

Question 8

Topological relationship between compartments.

Answer 8

Extracellular space connects with lumen of
ER, Golgi, vesicles, perinuclear space 

Cytosol connects with inside of the nucleus

Question 9

what is gated transport?

what does it rely on?

Answer 9

Gated transport is a regulated cellular mechanism that facilitates the movement of proteins and other molecules between topologically equivalent spaces, such as the nucleus and the cytoplasm, without the need for these substances to cross lipid membranes.

It relies on the specific recognition of signal sequences by receptor proteins that facilitate the transit through nuclear pore complexes, allowing for controlled and bidirectional exchange of vital cellular components.

Question 10

what the purpose of gated transport?

Answer 10

-Plays a crucial role in maintaining cellular function and responding to environmental signals.
- Important for the transport of larger macromolecules, like proteins and ribonucleoproteins, which cannot pass freely through lipid bilayers.

Answer 11

Nuclear Import Receptors
- soluble cytosolic proteins
- recognise nuclear localisation sequences (signal sequence directing protein to nucleus)
- deliver proteins to nuclear pore for transport

Nuclear Import Receptors (= reverse)
- nuclear export signal sequence
- nuclear export receptors (Bind to: Proteins with nuclear export signals.
Function: Transport these proteins out of the nucleus.)

Question 12

How does size affect transport across nuclear membrane.

Answer 12

Small molecules (<60kDa) can diffuse
Large molecules need active transport
DNA/RNA polymerases (100-200kDa)
Ribosomal subunits (30nm)

Question 13

what is Transmembrane Transport

Answer 13

Transmembrane transport is the movement of substances across cellular membranes, using specialized proteins known as membrane-bound translocators.
Is crucial for the import of proteins into organelles such as mitochondria and the endoplasmic reticulum (ER).

Question 14

what happens during transmembrane transport ?

Answer 14

During transmembrane transport:
-Unfolded proteins undergo transmembrane transport via translocators.
- Unfolded proteins navigate through these translocators, effectively “snaking” through the membrane.
-In mitochondrial import, proteins move from the cytosol into the mitochondria.
-In ER transport, proteins travel from the cytosol into the endoplasmic reticulum.

Answer 15

Soluble proteins
-transported fully across the membrane, particularly the ER membrane in eukaryotic cells, destined for secretion from the cell or to delivered to the lumen of the ER (OR inside other organelles)

Transmembrane proteins
-partly transferred across the membrane
remain embedded in membrane
destined to reside in a cellular membrane

Question 16

examples of soluble and transmembrane proteins.

Answer 16

soluble- hemoglobin /insulin/cyclin
transmembrane- glycoproetein hormone receptors/ ion channels/ transport proteins.

Question 17

Transport into the ER

Answer 17

-Co-translational
-N-terminal signal sequence (made first)
-Signal sequence recognized by signal
recognition particle (SRP)
-SRP binds to SRP receptor on ER membrane
-Polyribosomes attracted to ER (RER)
-Complex brought to translocator
-SRP-SRP receptor release
-Translocator transfers growing polypeptide chain through the membrane

Question 18

Vesicular transport. what is it for and what does it involve. what does the process rely on? what are the key pathways ?

Answer 18

FOR THE EXPORT OF PROTEINS FROM THE CELL.
- involves the transport of proteins within membrane-bound vesicles.
- These “membrane bags” encapsulate proteins, with the proteins remaining in the lumen of the vesicle.
- The process relies on the mechanisms of membrane budding and fusion.
- Key pathways include transport from the endoplasmic reticulum (ER) to the Golgi apparatus, from the Golgi to secretory vesicles, and ultimately from Golgi or vesicles to the cell surface.

Question 19

Other than transport of molecules, what are purposes of vesicular transport in the cell.

Answer 19

• Endocytosis
• Exocytosis
• Recycling of membrane components ( e.g. helps in retrieving membrane proteins and lipids from the plasma membrane after endocytosis)
• Cellular communication- vesicles carry and deliver signalling molecules.
• lysosomal function- essential for transporting enzymes to lysosomes, where cellular waste and damaged organelles are degraded.
• Homeostasis- they regulating levels of various substances within the cell and its environment.

Question 20

what are the two categories of signal Sequences?

Answer 20

Organelles:
Proteins that are destined for organelles typically have specific signal sequences that guide their transport.
Examples include:
Mitochondrial Targeting Sequence: Directs proteins to the mitochondria.
Nuclear Localization Signal (NLS): Directs proteins to the nucleus.

Secreted Proteins:
Proteins that are intended to be secreted outside of the cell contain an ER signal sequence that directs their translation and translocation into the ER. Once inside the ER, these proteins can be modified (such as glycosylation), folded, and then packaged into vesicles for transport to the Golgi apparatus and eventually to the cell plasma membrane for secretion.
Example of secreted proteins include hormones and digestive enzymes.

Question 21

How does cell signalling work?

Question 22

Continuous vs Discontinuous signal sequences.

Answer 22

Continuous:
- signal sequence made up of a singular, uninterrupted stretch of amino acids that serves a specific function. E.g. A hydrophobic stretch that is recognised by the signal recognition particle (SRP) during translation of proteins destined for the ER.

Discontinuous:
- signal sequences that are separated by other sequences or structures in the protein.
- recognition requires specific conformational changes or interactions with other molecules.

Question 23

Removal of signal sequences.

Answer 23

Post-Translational Modification: Once a protein with a signal sequence reaches its destination, the signal sequence is often cleaved off by specific enzymes (proteases). This process is essential for the maturation of the protein.

Examples: For instance, when proteins enter the rough ER, they typically have an N-terminal signal peptide that is recognized by a signal recognition particle (SRP) during translation. Once the protein is translocated into the ER lumen, the signal peptide is usually removed by the enzyme signal peptidase.

Question 24

Signal sequences in Final proteins.

Answer 24

May or may not be included :
In many cases, once the protein reaches its destined location and serves its role in the sorting process, the signal sequence is no longer necessary, and it is thus removed. Consequently, the final functional protein does not contain the signal sequence.

There are situations where signal sequences may remain part of the final protein, especially if they have a functional role within the protein’s activity or stability in its final location.

Question 25

Start and stop transfer signals are essential components of signal sequences in protein synthesis and translocation. Explain them further.

Answer 25

Start Transfer: The signal recognition particle (SRP) detects the emerging signal sequence of a nascent protein from the ribosome during translation, guiding the ribosome to the endoplasmic reticulum (ER) and inserting the sequence into the translocon for continued protein synthesis.

Stop Transfer: As the protein synthesizes, hydrophobic amino acids form a transmembrane domain. When these sequences are encountered, they trigger the stop transfer, allowing the hydrophobic segment to exit the translocon and embed in the membrane, typically adopting an α-helical structure.

Transmembrane Domain: The resulting hydrophobic (avoid water) segments, which form membrane-spanning α-helices, establish the protein’s topology (arrangement of the transmembrane segments), influencing how many times it crosses the membrane and its functional orientation within the cell.

Question 26

Secretary pathway

Answer 26

1) Co-translational trans-membrane transport into the ER
- The ribosome attaches to the ER membrane, and during translation, the growing polypeptide chain is threaded into the ER lumen through a translocation channel.

2) Exit from the ER and entry into the Golgi.
-Once the proteins are fully synthesized in the ER, they undergo proper folding and post-translational modifications (such as glycosylation).
-Properly folded proteins are then packaged into transport vesicles that bud off from the ER membrane. These vesicles transport the proteins to the Golgi apparatus, a key organelle for modifying and sorting proteins.

3)Travel through the golgi
-The proteins enter the Golgi from the ER at the cis face (the side closest to the ER) and travel through the stacked Golgi cisternae (flattened membrane sacs).
-As proteins pass through the Golgi, they undergo further modifications, such as additional glycosylation, phosphorylation, and sorting.

4) Exit from the Golgi via secretory vesicle
-Once the proteins are fully modified and sorted within the Golgi, they are packaged into secretory vesicles. These vesicles bud off from the trans face of the Golgi and contain the proteins to be secreted.

5) Secretion/exocytosis - fusion of the secretory vesicle with the plasma membrane.
- The secretory vesicles travel through the cytoplasm towards the plasma membrane. Upon reaching the membrane, vesicles undergo a process called exocytosis- where the secretory vesicle fuses with the plasma membrane, releasing its contents (the secretory proteins) into the extracellular space.

Question 27

Changes to protein structure during secretion- N-linked glycosylation.
what is its?
Where does it happen?
structural impact on proteins?

Answer 27

This occurs when a sugar molecule is attached to the nitrogen atom of an asparagine amino acid in a protein.

N-linked glycosylation starts in the ER where new proteins are being made. The addition of this sugar chain helps proteins fold correctly.

Structural Impact: The sugar chain modifies the protein’s shape, enhancing its stability and preventing it from sticking to itself (aggregation).

Chaperone proteins in the ER assist in ensuring that only properly folded proteins move on to the next steps.

Question 28

Changes to protein structure during secretion- O-linked glycosylation to the golgi apparatus.
what is it ?
structural impact on protein?

Answer 28

This involves the attachment of sugars to the oxygen atom of serine or threonine residues in proteins.

happens once the protein is in the golgi apparatus.

structural impact: O-linked glycosylation further alters protein structure, impacting stability, solubility, and how proteins interact with other molecules and receptors/ O-linked glycosylation in the Golgi enhances structural modifications that influence protein function and destination.

Question 29

Changes to protein structure during secretion- ER chaperones.

Answer 29

Ensure Quality Control: Chaperones recognize misfolded proteins and promote their refolding. If proteins cannot be correctly folded, they are targeted for degradation through the ER-associated degradation (ERAD) pathway, ensuring that only properly folded proteins proceed to the next stage of secretion.

Question 30

How do cells bring material in?

Answer 30

Relies on vesicles.
Two main types
Drinking (pinocytosis)
- Vesicles <150nm
- Receptor-mediated endocytosis

Eating (phagocytosis)
- Vesicles >250nm
- Specialised cells eat large particles

Question 31

summary of exocytosis:

Answer 31

• from ER to golgi to PM
• Signal sequence dictates path
• Chaperones check quality
• Sugar modifications made on journey through ER and Golgi
• Vesicular transport transfers proteins
• Vesicles are protein coated
• Membrane fusion deposits protein cargo

Question 32

summary of exocytosis

Answer 32

-PM to EE to LE to lysosome
- PM to EE to RE to PM
- Uptake of membrane & other cargo
- Pinocytosis/Receptor mediated endocytosis
- Phagocytosis (specialised)
- Lysosomes degrade ingested material

Question 33

what are the main differences between exocytosis and endocytosis.

Question 34

what are the key proteins in vesicular trafficking.

Answer 34

Coat proteins
Rab GTPases
SNAREs

Question 35

Characteristic and Functions of coated proteins.

Answer 35

Membrane Shaping:
The protein coat is primarily responsible for shaping the membrane into a cage-like structure. This shaping is essential for the budding of vesicles from donor membranes, as it facilitates the formation of a spherical vesicle that can separate from the membrane.

Capture of Molecules:
Coated proteins aid in the selection and capture of specific molecules that need to be transported. By clustering receptors and their corresponding cargo (specific) , these proteins ensure that the vesicle carries the right contents toward its destination, thus playing a central role in maintaining cellular function and organization.

Reversible Assembly:
Coated proteins can assemble and disassemble in response to cellular signalling, allowing for dynamic changes in vesicle formation.

Question 36

In summary whats the function of coated proteins?

Answer 36

Coated proteins are proteins that form a coat around vesicles and play a crucial role in the transport of cargo within cells. These proteins help in budding, shaping the vesicle, and selecting the proper cargo to be transported.

Question 37

what are the major components of coated proteins?

Answer 37

Clathrin
COPI and COPII

Question 38

Clathrin- structure AND function

Answer 38

Structure:
forms a basket-like structure around vesicles that bud off from cell membranes. It consists of three heavy chains and three light chains that assemble to for a triskelion shape.

Function:
Clathrin plays a crucial role in endocytosis—Specifically, clathrin-mediated endocytosis is responsible for:
1) Pinching Off Vesicles: Clathrin coats the cytoplasmic side of the membrane, facilitating the invagination (BENDING) of the membrane to form a vesicle.

2) Transporting Cargo: Clathrin-coated vesicles transport various cargo, including cholesterol, growth factors, and neurotransmitters.

3) Selective Uptake: Clathrin works alongside adaptor proteins (APs), which bind to specific receptors and cargo molecules on the inner surface of the membrane, ensuring the vesicle captures the desired cargo.

4) Vesicle Traffic: Clathrin-coated vesicles typically transport materials from the plasma membrane to early endosomes and then onwards to lysosomes or other destinations within the cell.

Question 39

Clathrin - Origin and Destination

Answer 39

Clathrin + adaptin 1 (AP-1)
Originate from Golgi apparatus -they bud from the TRANS-GOLGI NETWORK.
Destination: Once these vesicles are formed, they can either fuse with early endosomes to deliver their contents to LYSOSOMES for degradation and recycling (back to the membrane)

Clathrin + adaptin 2 (AP-2)
origin: primarily associated with the PLASMA MEMBRANE and is involved in the endocytic pathway.
Destination: facilitate the internalization of membrane proteins and other substances from the extracellular environment into EARLY ENDOSOMES .

Question 40

Defintition of ENDOSOSMES

Answer 40

membrane- bound vesicles that regulate the trafficking of proteins and lipids in eukaryotic cells.

Question 41

COPI (coat protein complex I)- structure and function.

Answer 41

stucture: COPI is composed of seven distinct subunits. Forms a cage-like structure that wraps around vesicles, aiding in their budding from membranes.

Function:
1) Vesicle Transport: COPI is primarily responsible for retrograde transport, moving proteins from the Golgi apparatus back to the ER and between Golgi compartments.

2) Cargo Selection: It recognizes and binds specific cargo proteins that carry retrieval signals, ensuring that only the correct proteins are transported back to the ER for reuse or modification.

Question 42

COPII- structure and function

Answer 42

Structure : COPII is composed of five core proteins (sar1, sec23, sec24, sec13,sec31).Cage-like Structure: The COPII complex assembles into a spherical coat that wraps around vesicles budding from the endoplasmic reticulum (ER), facilitating membrane curvature and vesicle formation.

Function:
Vesicle Budding: COPII mediates the transport of proteins from the ER to the Golgi apparatus. It promotes the budding of vesicles from ER exit sites.

Cargo Selection: The complex recognizes and selectively packages proteins destined for secretion or further modification in the Golgi. It binds to proteins that contain specific exit signals.

Question 43

Origin and destination of COP proteins.

Answer 43

ER to golgi apparatus (MAIN ONE)
Golgi cisterna to golgi cisterna ???
Golgi appartus to ER.

Question 44

Rab-GTPases.

Answer 44

Rab GTPases are a large family of small GTP-binding proteins that play crucial roles in intracellular vesicle trafficking and membrane dynamics. Characterised by their ability to bind and hydrolze guanosine triphosphate (GTP) to GDP (guanosine diphosphate).

Question 45

what are the 4 cellular functions of Gap GTPases.

Answer 45

1) Specificity in Vesicular Transport:
Rab GTPases are crucial for directing vesicles to their correct destinations within the cell. Each Rab protein is associated with specific organelles or compartments, which helps in maintaining the fidelity of intracellular transport.

2) Tethering of Membranes:
Rab GTPases function through their interactions with Rab effectors, which facilitate the tethering of vesicles to target membranes. This tethering represents the first connection between donor and target compartments, setting the stage for subsequent fusion. (Rab proteins can link membranes that are more than 200 nm apart, effectively bridging significant distances within the cellular environment)

Collaboration with SNAREs: Rab GTPases work in conjunction with SNARE proteins to facilitate membrane fusion. While Rab proteins ensure that the right vesicle arrives at the correct target, SNAREs mediate the actual fusion process. This collaboration is essential for the delivery of cargo molecules, such as proteins and lipids, to their intended locations.

Coordination of Transport Processes: By coordinating the actions of Rab effectors and SNAREs, Rab GTPases play a critical role in the overall orchestration of vesicular trafficking, ensuring that materials are transported efficiently and accurately within the cell.

Question 46

what are the implications of Rap GTPases.

Answer 46

Dysfunction in Rab GTPases is linked to diseases like neurological disorders, cancer, and infections, highlighting their potential as therapeutic targets.

Question 47

Activation and inactivation of Rab GTPases

Answer 47

Activation: Rab GTPases are activated when they bind to GTP. This typically involves the action of guanine nucleotide exchange factors (GEFs), which facilitate the exchange of GDP for GTP.

Inactivation: After their role in vesicle trafficking is completed, Rab proteins are inactivated by hydrolyzing GTP to GDP, often with the aid of GTPase-activating proteins (GAPs).

Question 48

SNAREs. what is it ?

Answer 48

SNAREs (Soluble N-ethylmaleimide-sensitive factor Attachment Receptors) are a family of proteins. essential for the fusion of vesicles with target membranes in intracellular trafficking. Vesicle fusion relies on SNAREs to ensure accurate and efficient membrane merging.

Question 49

what are the two types of SNAREs?

Answer 49

v-SNAREs (vesicle SNAREs): Located on the vesicle membrane, they identify and bind to the appropriate target.

t-SNAREs (target SNAREs): Found on the target membrane, they interact with v-SNAREs to facilitate fusion.

Question 50

SNAREs . what mechanism of action is involved and what’s the importance of this . interaction.

Answer 50

When a vesicle moves along cytoskeletal fibers from its donor compartment (ORIGIN) to its target compartment(DESTINATION), it carries a specific v-SNARE protein on its membrane. When it the reaches the target compartment, the v-SNARE interacts with a complementary t-SNARE on the target membrane.

importance: ensuring specificity in vesicular transport.

Question 51

Explain how winding and fusion of SNAREs works.

Answer 51

Winding and Fusion: The SNARE proteins wind around each other, creating a tight complex. This winding action pulls the two membranes closer together, leading to a fusion process where the lipid bilayers merge.

Question 52

SNAREs - Role in Cellular Processes.

Answer 52

neurotransmitter release in neurons,
hormone secretion,
the transport of proteins and lipids within the cell.