6. The Golgi apparatus and membrane traffic

Question 1

Membrane traffic - overview

Answer 1

•The proteins contained in the plasma membrane (PM) are the primary
mechanism by which the cell senses the environment
•The composition of the PM (both proteins and other components) must
constantly change in response to the environment
•Proteins that need to be secreted must pass through the PM.
•PM is altered by two main processes: Endocytosis + Exocytosis

Question 2

Endocytosis

Answer 2

– describes the removal of PM components into endosomes for delivery/destruction

Question 3

Exocytosis

Answer 3

– describes the delivery of proteins/carbohydrates and lipids to the PM/Extracellular environment

Question 4

Intravesicular trafficking

Answer 4

• Endocytosis + Exocytosis rely on the formation of transport vesicles (TVs) containing cargo that are then migrate to the correct compartment and release contents by fusion – intravesicular trafficking

Cytosol with the lumen acts as the donour compartment -> binding and fusion occurs -> Target compartment

• Intravesicular trafficking occurs between a number of organelles: the Endoplasmic reticulum, Golgi Complex, Endosomal and Lysosomal compartments.
• Vesicles are formed by the interaction of coat proteins with specific membrane regions

Coat assembly and cargo selection -> Bud formation -> vesicle formation -> uncoating

Question 5

Clathrin movie

Answer 5

• TVs are recognised by the type of coat they have: 3 main types COP1 = coat protein 1; COPII – coat protein II (anterograde)

Targeting is controlled by Rab-GTP proteins, fusion via SNAR

• SNAP (Soluble NSF Attachment Protein) Receptor)
• NSF N-ethylmaleimide sensitive fusion proteins or
  N-ethylmaleimide sensitive factor

v-SNARES + Rab-GTP -> (Tethering) -> Rab effector (tethering protein) + t-SNARES -> (Docking + Fusion)

Question 6

Interaction of v-SNARE and t-SNARE leading to membrane fusion

Answer 6

synaptic vesicle with v-SNARE (synaptobrevin) + t-SNARE (symtaxin) + t-SNARE (Snap-25)

H20 -> form Stalk -> Hemifusion (fusion of 2 membranes into one) -> Fusion

Question 7

Golgi complex - functions

Answer 7

Modifies proteins and lipids from the ER and distributes them to the plasma membrane (PM), Endosomes and Secretory vesicles –‘cellular post office’

Interacts with endocytic pathway via endosomes

Major site for carbohydrate synthesis (glycosoaminoglycans synthesised here)

Oligosaccharide side chains attached to proteins

Question 8

Golgi complex - structure + location

Answer 8

Nuclear membrane:
=> Cis golgi network (CGN) with golgi vesicles
-> cis cisterma
-> medial cisterna
-> trans cisterma
=> trans golgi network (TGN)

Question 9

Golgi complex - Transport of contents from the ER to the golgi

Answer 9

Endoplasmatic reticulum -> Golgi microtubule

ER -> COPII coat -> motor protein -> retrieval transport of COPI coat from the vesicular tubular cluster -> cis golgi network

Question 10

Golgi complex - transport of material from the ER to golgi

Answer 10

• Retrieval of soluble ER-located proteins is achieved by vesicular transport
• Typified by KDEL receptor that binds to such proteins and carries them to ER in COPI coated TV

=> ER:

• > secretory protein
• > KDEL receptor protein
• > soluble ER resident protein

Forward pathway and retrieval pathway -> COPI coat attached to vesicular tubular cluster

=> cis golgi network
=> golgi stack
=> trans golgi network

Question 11

Golgi complex - function

Answer 11

Oligosaccharide processing/Glycosylation of proteins

• Built up sequentially by specific enzymes
• N-linked glycosylation
• promotes protein folding
• increases solubility
• targets protein for chaperone
• increases resistance to enzymatic digestion
• O-linked glycosylation important in mucin/proteoglycan
formation
• specificity of receptor signalling (Notch)

• Oligosaccharide processing:
• Ordered pathway for oligosaccharide processing in Golg

ER lumen:

• > glucosidase I
• > glucosidase II
• > ER mannosidase

Golgi lumen: (endo H-sensitive)
=> high-mannrose
- golgi mannosidase I
- N-acetylglucosamine transferase I

-> Golgi mannosidase II

=> complex oligosaccharide (endo H-resistant)
- ASN

Question 12

Golgi Complex Function - Oligosaccharide processin

Answer 12

• Prominent in secretory cells
• Occurs in order from cis to trans, evidence from localisation studies

-> Endoplasmatic reticulum

=> Cis golgi network:
Sorting - phosphorylation of oligosaccharides on lysosomal proteins

=> cis cisterma: (golgi stack)
removal of man

=> medial cisterma: (golgi stack)
removal of man + addition of GicNac

=> trans cisterma: (golgi stack)
addition of golgi + addition of NANA

=> trans golgi network
Sorting - sulfation of tyrosines + carbohydrates

• > lysosome
• > plasma membrane
• > secretory vesicle

Two main types of N-linked oligosaccharides: complex and high-mannos

A) Core region
B) complex oligosaccharide
C) high-mannose oligosaccharide

Question 13

Golgi Complex Function: Proteoglycan

assembly

Answer 13

-