Protein Trafficking & Secretion

Question 1

Learning Outcomes

Answer 1

• Outline how proteins with distinct localisation signals are
  delivered to the nucleus and mitochondrion.
• Be able to list the steps involved producing a secreted protein.
• Outline the functions of vesicles.
• Identify the roles of coat proteins, Rabs, and SNAREs.
• Describe the difference between regulated and unregulated
  exocytosis in secretion.

Question 2

You already know some of this…

Answer 2

• Cells have many membranous
  organelles that regulate
  protein transport (Yu Suk Choi’s
  lectures)
• Translation produces
  peptides/proteins from
  ribosomes within the
  cytoplasm/Rough ER (Mitali
  Sarkar-Tyson’s lectures)

Question 2

You already know some of this…

Answer 2

• Biochemical messengers are
  secreted from cells
• Nuclear receptors translocate
  from cytoplasm → nucleus

Question 3

And, very briefly, …

Answer 3

• Secreted proteins are
  transcribed in the nucleus,
  translated on ribosomes,
  often at the rough ER,
  modified by the Golgi
  apparatus, packaged into
  vesicles and exocytosed in
  response to stimulus

Question 4

So……
How do the&raquo_space;>30,000
proteins find their
correct cellular locations?

Answer 4

Proteins are directed to their destinations
via signals in the amino acid sequence

Question 4

How can proteins be delivered to their
appropriate destinations?

Answer 4

Question 5

Proteins are produced by ribosomes and
function in many different locations

Answer 5

Question 6

Protein Destinations → nucleus

Answer 6

• Nuclear localization signal (NLS) anywhere in protein
• Translation in cytoplasm, NLS not removed
• import via nuclear pore complex (NPC)

Question 6

Protein Destinations → nucleus

Answer 6

Question 7

Import & Export via Nuclear Pore Complexes (NPCs)

Answer 7

• NPCs span both layers of nuclear envelope
• Protein NLS binds a nuclear import receptor
  (importin)
• Complex binds NPC proteins → import

Question 8

Import & Export via Nuclear Pore Complexes (NPCs)

Answer 8

• NPCs span both layers of nuclear envelope
• Protein NLS binds a nuclear import receptor
  (importin)
• Complex binds NPC proteins → import
• Ran-GTP binds complex → complex dissociates
  → protein delivered
• Importin is recycled (control of import)

Question 9

Import & Export via Nuclear Pore Complexes (NPCs)

Answer 9

Question 10

Import & Export via Nuclear Pore Complexes (NPCs)

Answer 10

• NPCs span both layers of nuclear envelope
• Protein NLS binds a nuclear import receptor
  (importin)
• Complex binds NPC proteins → import
• Ran-GTP binds complex → complex dissociates
  → protein delivered
• Importin is recycled (control of import)
• Importin-Ran-GTP complex leaves nucleus →
  GAP hydrolises GTP→GDP → dissociates
• Importin is now free to import proteins again!

Question 10

Import & Export via Nuclear Pore Complexes (NPCs)

Answer 10

Question 11

Import & Export via Nuclear Pore Complexes (NPCs)

Answer 11

Question 11

Import & Export via Nuclear Pore Complexes (NPCs)

Answer 11

• NPCs span both layers of nuclear envelope
• Protein NLS binds a nuclear import receptor
  (importin)
• Complex binds NPC proteins → import
• Ran-GTP binds complex → complex dissociates
  → protein delivered
• Importin is recycled (control of import)
• Importin-Ran-GTP complex leaves nucleus →
  GAP hydrolises GTP→GDP → dissociates
• Importin is now free to import proteins again!
• Nuclear export involves a NES, nuclear export
  receptor, and Ran-GTP-binding (GTP→GDP
  hydrolysis)

Question 12

Protein Destinations: mitochondria (or plastid)

Answer 12

• Produced as mitochondrial precursor proteins
• N-terminal localization signal (translated first)
• Translation in cytoplasm, signal removed after import
• Folding structure blocked by chaperones (hsp70)
• Import via TOM/TIM complex

Question 12

Translocation into Mitochondria is via TOM/TIM
Protein Translocators

Answer 12

• TOM: transporter outer membrane complex
• TIM: transporter inner membrane complex
• SAM: sorting and assembly machinery
• inserts proteins into outer membrane
• OXA: oxidase activity
• inserts proteins into inner membrane

Question 12

Import & Export via Nuclear Pore Complexes (NPCs)

Answer 12

Question 13

Translocation into Mitochondria is via TOM/TIM
Protein Translocators

Answer 13

Import to matrix space
* Protein signal binds receptor protein in TOM
* Signal region translocates to TIM
* ATP hydrolysis drives import, requires [H+
]
gradient (supplied by ETC)
* Signal peptide cleaved → mature protein

Question 13

Translocation into Mitochondria is via TOM/TIM
Protein Translocators

Answer 13

Question 14

Translocation into Mitochondria is via TOM/TIM
Protein Translocators

Answer 14

Question 15

Protein Destinations: secretion or membrane

Answer 15

• N-terminal ER signal sequence (cleaved inside ER)
• Co-translational import
• begins in cytoplasm → signal peptide/ribosome → rough ER
• Shuttled to Golgi apparatus
• Shuttled to membrane → inserted/secreted

Question 16

Vesicles are
not simply
passive sacks!

Answer 16

Question 16

ER translation/translocation

Answer 16

* Nascent signal peptide binds signal recognition particle (SRP) * SRP pauses translation, targets ribosome-growing peptide to SRP receptors on ER * Inserts ribosome-peptide into translocator, translation continues

Question 17

ER translation/translocation

Answer 17

Question 18

Proteins are packed into vesicles for trafficking & secretion

Answer 18

Question 19

Vesicles are assembled by coat proteins

Answer 19

* Required for formation * Discarded before fusion * Clathrin * Golgi plasma membrane * COPI (coat protein 1) * Golgi → Golgi * Golgi → ER * COPII (coat protein 2) * ER → Golgi

Question 20

Vesicles are assembled by coat proteins

Answer 20

Question 20

Rabs and SNAREs mediate docking & fusion

Answer 20

Question 20

Rabs and SNAREs mediate docking & fusion

Answer 20

* Rabs give the address * Rabs bind Rab effectors → motor/tether to membrane * SNAREs mediate fusion * v-SNAREs on vesicle * t-SNAREs on target membrane

Answer 20

Question 21

Golgi apparatus

Answer 21

Question 21

Golgi apparatus

Answer 21

* cis-Golgi receives and sorts all ER proteins * Post-translational modification of proteins for secretion (glycosylation, sulfation) * Modified vesicles bud off from trans-Golgi surface

Question 22

Exocytosis can be constitutive or regulated

Answer 22

Question 23

Neurotransmission – regulated secretion

Answer 23

Question 24

Summary

Answer 24

Question 25

Summary

Answer 25

* Proteins are targeted to their subcellular location by localisation signal peptides that interact with specific transport molecules. * Secreted proteins are translated at the RER, modified in the Golgi apparatus and assembled into vesicles. * Coat proteins enable packaging of vesicles, Rab proteins target vesicles to a specific destination, and SNAREs facilitate docking and fusion of vesicles. * Regulated exocytosis involves the production of secretory vesicles, their storage, and release in response to signal.