The Endomembrane System

Question 1

How do prokaryotes secrete proteins

Answer 1

Through signal peptides (usually at the N-terminus) that are ~20 hydrophobic amino acids long and often preceded by positively charged residues. These peptides guide secretion and are usually cleaved post-translocation

Question 2

What is the role of the SRP in prokaryotic secretion

Answer 2

1. SRP (Signal Recognition Particle) recognizes the signal peptide, arresting translation.
2. SRP directs the ribosome to a translocation channel.
3. SRP dissociates, translation resumes, and the protein is co-translationally translocated.
4. The signal peptide is cleaved by a protease after translocation

Question 3

How are hydrophobic stretches of proteins interpreted during synthesis

Answer 3

They are interpreted by the translocation channel as start-transfer or stop-transfer signals, guiding insertion into membranes

Question 4

Why are prokaryotic cells often small and rod shaped

Answer 4

Their single membrane must fulfill all functions, and SA:V constraints limit their size. Small, rod-shaped cells maintain efficient diffusion and energy/nutrient exchange

Question 5

What is the endomembrane system and its function

Answer 5

A collection of membranous organelles (ER, Golgi, vesicles, lysosomes) that handle synthesis, transport, and degradation of proteins/lipids. It increases membrane surface area and creates compartmentalised microenvironments

Question 6

What is the origin of endomembrane organelles

Answer 6

They are derived endogenously from the plasma membrane

Question 7

What are key roles of the plasma membrane

Answer 7

Interacts with the external environment, handles material exchange, and serves as the cell boundary

Question 8

How are macromolecules imported/exported across the nuclear envelope

Answer 8

Via nuclear pore complexes, which are selective, ATP-dependent channels

Question 9

What happens to secreted and membrane proteins in the ER

Answer 9

They enter the rough ER, where translation and translocation occur. Proteins are folded and glycosylated (N-linked glycans on Asn residues)

Question 10

What is the difference between rough and smooth ER

Answer 10

Rough ER: ribosomes attached; site of protein synthesis/folding.
Smooth ER: lacks ribosomes; synthesises lipids and sterols

Question 11

How are proteins transported from the ER to the Golgi

Answer 11

In COP-II vesicles, which bud from the ER and carry only correctly folded proteins

Question 12

What are the key roles of the Golgi apparatus

Answer 12

1. Trimming and processing N-linked glycans.
2. Synthesising glycolipids and sphingolipids.
3. Functionally compartmentalised into cis, medial, and trans cisternae for sequential processing.

Question 13

Is a signal required for ER export

Answer 13

No specific signal is required - bulk flow can export proteins by default. However, signals are required to retain proteins in the ER

Question 14

What are ER retention/retrieval signals

Answer 14

Short amino acid sequences (e.g. KDEL for soluble proteins, KKXX for membrane proteins) that are both necessary and sufficient to retain proteins in the ER

Question 15

How are ER resident proteins retrieved

Answer 15

Via COP-I vesicles, which recognize retrieval signals and return proteins from the Golgi to the ER

Question 16

How are Golgi-resident proteins localised

Answer 16

Through their transmembrane domain (TMD) length and cytosolic tail, which match the Golgi membrane thickness and signal localisation

Question 17

How do membrane thicknesses differ across organelles

Answer 17

Plasma Membrane > Golgi > ER
Proteins match their TMD length to the membrane thickness for proper localisation

Question 18

What are the four fates of proteins in the Golgi

Answer 18

1. PM/secretion (default)
2. Lysosome via CCV (signalled)
3. Return to ER via COP-I (signalled)
4. Retention in Golgi (signalled)

Question 19

What is the function of the endocytic pathway

Answer 19

Imports macromolecules from the environment to lysosomes or vacuoles for digestion

Question 20

What are the main internalisation mechanisms

Answer 20

1. Phagocytosis (engulfment of large particles)
2. Endocytosis (small vesicle internalisation)

Question 21

How are receptors and cargo handled in endocytosis

Answer 21

Receptor and cargo bind at the PM and are internalised.
Travel to early endosome (EE).2
Receptors are recycled; cargo progresses to late endosome (LE) and lysosome.

Question 22

What are CCVs and how are they formed

Answer 22

Vesicles with two layers:
1. Clathrin (outer): bends membrane.
2. Adaptin (inner): selects cargo via receptor tails

Question 23

How do LDL particles enter cells

Answer 23

Via LDL receptors into clathrin-coated pits, forming CCVs that go to the early endosome where LDL is released

Question 24

How are lysosomal hydrolases made and targeted

Answer 24

Made in the ER as inactive pro-enzymes.

Modified in the cis-Golgi (mannose-6-P tag via signal patch).

Sorted by M6P receptor into clathrin-coated vesicles with AP1 adaptin to go to the late endosome.

Question 25

What happens to lysosomal hydrolases in the late endosome

Answer 25

M6P receptor releases cargo due to low pH. Hydrolases become active via proteolytic cleavage in the lysosome

Question 26

How does digestion differ between prokaryotes and eukaryotes

Answer 26

Prokaryotes: Secrete enzymes for external digestion, then import products. Eukaryotes: Internalise macromolecules via endocytosis and digest internally in lysosomes

Question 27

What determines whether a protein remains in the ER or is secreted

Answer 27

ER-resident proteins have specific retrieval signals (e.g. KDEL or KKXX) that are necessary and sufficient to retain them; without the signal, they are secreted by default

Question 28

What is the role of COP-II vesicles

Answer 28

COP-II vesicles mediate export from the ER to the Golgi, carrying properly folded proteins and lipids

Question 29

How does membrane thickness relate to protein localisation

Answer 29

The increasing thickness from ER → Golgi → PM correlates with increasing TMD length of membrane proteins, determining their localisation

Question 30

What is the functions of adaptins in the CCVs

Answer 30

Adaptins form the inner layer of CCVs and recognise cytoplasmic tails of cargo receptors, ensuring selective cargo loading

Question 31

What triggers cargo release from receptors in the endosome

Answer 31

A drop in pH in the early endosome causes conformational changes in receptors, leading to cargo release

Question 32

What is a 'signal patch' and what does it do

Answer 32

A signal patch is a 3D structural feature on lysosomal hydrolases that is recognised by cis-Golgi enzymes, which then phosphorylate a mannose residue, targeting the protein for lysosomal delivery

Question 33

What is the role of the NSF complex in vesicle fusion

Answer 33

NSF disassembles SNARE complexes after vesicle fusion, allowing SNAREs to be reused in future trafficking steps

Question 34

What is the function of the Golgi's trans face

Answer 34

The trans face is the exit side of the Golgi, where proteins are sorted and packaged for delivery to the PM, lysosomes, or back to the ER

Question 35

How are Golgi-resident enzymes retained in the Golgi

Answer 35

The trans face is the exit side of the Golgi, where proteins are sorted and packaged for delivery to the PM, lysosomes, or back to the ER

Question 36

Why is the lumen of the ER considered topologically equivalent to the extracellular space

Answer 36

Because proteins in the ER lumen do not need to cross another membrane to be secreted - they are already in the same orientation as the extracellular environment

Question 37

What is the 'bulk flow' hypothesis for ER export

Answer 37

Most proteins don't require a specific export signal to leave the ER - they exit by default unless retained by a signal

Question 38

What experimental evidence supports the bulk flow model of ER export

Answer 38

Bacterial proteins lacking export signals are secreted when tagged with a eukaryotic signal peptide and expressed in eukaryotic cells, showing that export doesn't require a specific signal

Question 39

How does membrane lipid composition affect organelle identity

Answer 39

Differences in lipid composition affect membrane thickness, which in turn influences where membrane proteins with different TMD lengths localise (e.g. PM > Golgi > ER)

Question 40

How is TMD length used to localise proteins to specific organelles

Answer 40

TMD length is matched to membrane thickness - shorter TMDs stay in thinner membranes like the ER, while longer ones are sorted to thicker membranes like the PM

Question 41

What is the structure and function of the clathrin lattice

Answer 41

Made of clathrin triskelions, it forms a stable, curved cage-like lattice that drives vesicle formation and curvature at the PM and Golgi

Question 42

How does AP1 and AP2 adaptin complexes differ

Answer 42

AP1 operates at the trans-Golgi for lysosomal targeting, recognising Man-6P receptors; AP2 functions at the PM, helping form endocytic vesicles (e.g. for LDL uptake)

Question 43

How are Mannose-6-Phopahte

Answer 43

They release cargo in the late endosome and are recycled back to the trans-Golgi via transport vesicles

Question 44

How does receptor-mediated endocytosis ensure specificity

Answer 44

Receptors bind specific ligands at the PM, concentrate into coated pits, and are internalised via CCVs, ensuring only bound molecules enter the cell