lecture 7

Question 1

What sorts of compartments are there within a cell?

Answer 1

• nucleus surrounded by nuclear membrane
• continuous with endoplasmic reticulum
• golgi apparatus
• lysosome
• endosome
• peroxisome
• mitochondria

Question 2

What are the topological relationships between compartments in a eukaryotic cell?

Answer 2

• the plasma membrane separates the inside of the cell from the outside
• but not quite this simple
• the inside of the organelles is more similar to the outside of the cell
• not completely true that e.g. the inside of the golgi apparatus is exactly the same as outside the cell but whatever was on the outer surface of the plasma membrane will be on the inner surface of the organelles

Question 3

What are the three basic types of protein trafficking?

Answer 3

• gated transport: in and out of the nucleus
• transmembrane transport: transports proteins into peroxisomes, mitochondria, plastids, and the ER
• vesicular transport: transport between vesicles (ER, Golgi, secretory, late endosome, lysosome, early endosome, cell exterior)
• the first forms of transport are very individual while vesicular transport might involve the movement of many hundreds/thousands of proteins at once

Question 4

What directs proteins to their correct ‘address’ in the cell?

Answer 4

• signal sequences are like Post codes for proteins
  e.g. import into nucleus= pro-pro-lys-lys-lys-arg-lys-val
  while export from nucleus = leu- ala-leu-lys-leu-ala-gly-leu-asp-ile

Question 5

Describe the nuclear envelope

Answer 5

• the nucleus is surrounded by a double-membrane envelope that is penetrated by nuclear pores and is continuous with the endoplasmic reticulum

Question 6

What is the nuclear pore complex?

Answer 6

• the nuclear pore is not just a simple hole
• an intricate structure of various proteins and fibrils
• like a basketball hoop
• fibrils important for recognising proteins that enter the nucleus
• it is a gated diffusion barrier
• the limit size for free diffusion is around 60 kD (amino acids, ions, small organic molecules can freely diffuse through this pore)
• larger molecules can be actively transported through the NPC

Question 7

What are the nuclear import receptors?

Answer 7

• proteins that need to go into the nucleus (cargo proteins) bind to specific nuclear import receptors via nuclear localisation signals (NLS)
• nuclear import receptors interact with NPC proteins to transfer cargo in/out of the nucleus

Question 8

What gives directionality to nuclear transport?

Answer 8

Ran GTPase

• functions like a molecular switch
• ON in the nucleus, OFF in the cytosol
• Ran-GAP is located in the cytosol - assists the Ran-GTPase to hydrolyze the GTP back to GDP
• Ran-GEF is located in the nucleus - guanine exchange factor, assist in the exchange from GDP to GTP

Question 9

Describe the model of nuclear transport.

Answer 9

Import:

1. Cargo with NLS bind NIR
2. NIR shuttles into the nucleus via the NPC
3. Ran GTP binds to NIR to discharge the cargo
4. NIR shuttles out of the nucleus via the NPC
5. Ran-GAP activates Ran-GTP hydrolysis to form Ran-GDP in the cytosol
6. Ran-GDO actually has its own NIR to shuttle it back into the nucleus where it is converted to Ran-GTP by Ran-GEF

Export:

• Export of Cargo from the nucleus is similar except that:
• Ran-GTP promotes binding of cargo (with nuclear export signal) to Nuclear Export Receptor (NER/exportin)
• Ran-GDP dissociates cargo from the NER

For both export and import, cycling of the NIR and NER through the pore is independent of Ran-GTP and cargo
Ran-GTP provides directionality.

Question 10

What is the importance of the NLS?

Answer 10

Nuclear localisation requires an intact and functional NLS:

• normal Lys-rich sequence targets a protein to nucleus
• mutation of Lys->Thr causes cytoplasmic retention
• Add an NLS to a cytoplasmic protein -> nuclear localisation

Question 11

What are the particular mechanisms/complexes of different organelles in regards to transmembrane transport of proteins?

Answer 11

• mitochondria: TOM/TIM complexes
• chloroplasts - SRP-like, Translocators
• peroxisomes - Peroxins
• Endoplasmic reticulum: Ribosome, SRP/Sec61 translocators
• all of these require energy: usually ATP/GTP hydrolysis

Question 12

What does transmembrane transport mean?

Answer 12

• transport of a protein ACROSS a membrane

- NOT transport of ‘transmembrane proteins’

Question 13

How does transmembrane transport occur across a mitochondria?

Answer 13

• Cytosolic protein associated with small Hsp70 proteins keeping it in an unfolded state
• Binds to a receptor on the outer mitochondrial membrane that is part of the TOM complex
• With the addition of energy the Hsp70 proteins dissociate and the protein is fed through a translocator in the TOM complex
• then passess through the TIM complex on the inner mitochondrial membrane (this also requires energy)

Question 14

How does transmembrane transport occur across the endoplasmic reticulum?

Answer 14

• the act of getting proteins into the ER couples translation with their transport
• in the cytosol there are free ribosomes/polyribosomes which will translate proteins
• proteins that are being transported into the ER end up being translated by ribosomes bound to the ER
• before it is even its fully folded state it is fed through the translocator into the ER, where it then completes folding
• transport into the ER is the first step in getting proteins into other organelles - from here it goes via vesicular transport

Question 15

How do ER signal peptides and SRP direct ribosomes to the ER membrane?

Answer 15

• peptide synthesis is initiated on FREE ribosomes
• signal sequence of nascent polypeptide emerges from ribosome, binds an SRP (Signal Recognition Particle) and pauses translation
• SRP binds SRP receptor and directs ribosomes to the ER membrane
• Ribosome pore docks with translocator and SRP released
• Translation contintues into the ER lumen

Question 16

Describe the translocation of a soluble protein across the ER membrane.

Answer 16

• translocator consists of 4x Sec61 complexes
• pore opens when the ribosome (+nascent peptide) binds translocator and polypeptide feeds through
• the ER signal peptide acts as a “start-transfer” signal
• ER signal cleaved by peptidase, exists translocator and degraded
• mature protein deposited in the lumen

Question 17

How is a single-pass transmembrane protein integrated into the ER?

Answer 17

Similar to secreted protein BUT:

• 2nd stop-transfer sequence (hydrophobic) stops transfer but not synthesis
• carboxyl tail ends in the cytoplasm not ER lumen

Question 18

How is a double-pass membrane protein inserted into the plasma membrane?

Answer 18

• internal signal peptide binds translocator; the amino terminal is left in the cytoplasm and middle peptide region feeds through translocator
• 2nd stop-transfer signal stops translocation but not synthesis
• carboxyl tail ends in the cytoplasm but not ER lumen
• start-transfer not cleaved since internal

Question 19

Give an example of a multipass membrane protein

Answer 19

• some proteins have multiple transmembrane domains
• can be mapped by amino acid hydrophobicity plots
• rhodopsin - g protein - has 7 transmembrane domains

Question 20

What happens when a protein enters the ER?

Answer 20

• protein glycosylation occurs in rough ER as soon as proteins enter the lumen
• transfer of precursor oligosaccharide (glucose, mannose, N-acetylglucosamine) by transferase enzyme associated with translocator (oligosaccharyl transferase)

Question 21

What is vesicular transport?

Answer 21

Transport vesicles bud off from one compartment and fuse with another

Question 22

What is conversation of topology?

Answer 22

Vesicle contents are always separated from cytosol (inside vesicle = extracellular space)

Question 23

What are the three kinds of vesicular transport?

Answer 23

• biosynthetic secretory pathway
• endocytosis
• retrieval pathways

Question 24

What are the intracellular compartments in the eukaryotic cell involved in the biosynthetic-secretory and endocytic pathways?

Answer 24

(- nuclear envelope continuous with)

• endoplasmic reticulum
• golgi apparatus (cisternae)
• secretory vesicle
• lysosome
• late endosome
• early endosome
• plasma membrane

Question 25

What are coat proteins?

Answer 25

- transport vesicles bud off from coated regions of membranes - the coat assists in concentrating specific membrane proteins and forms the vesicle by causing curvature of the membrane - there are three coat proteins: clathrin (cell membrane, trans Golgi network), COPI (Golgi apparatus), and COPII (ER)

Question 26

Describe clathrin and its function

Answer 26

- clathrin forms 'clathrin-coated pits' on the plasma membrane (and trans golgi) which then forms the vesicles - three clathrin heavy chains and three clathrin light chains combine to form a structure called a triskelion - has a 3D strucutre which is what causes budding to occur - tips of the heavy chains bind to adaptor proteins which are bound to cargo membrane protein - forms a cage - the assembly of the coat induces curvature to the membrane - adaptins bind both clathrin triskelions and cargo receptors - once the vesicle is fully formed the clathrin molecules are released to form the naked transport vesicle - other proteins in the naked transport vesicle tell it where to go

Question 27

What is dynamin?

Answer 27

Dynamin is a GTPase that destabilises the bud neck so that the lipid bilayers flow together - blocked by some dynamin mutations

Question 28

How does a naked vesicle know where to go?

Answer 28

Rab proteins guide vesicle targeting and SNAREs mediate membrane fusion. Complementary sets of vesicle snare (v-snares) and target snares (t-snares) determine the selectivity of transport-vesicle docking Together these proteins cause the vesicle to dock at the correct location and then fuse with the membrane. Rab proteins associate with Rab effector (tethering protein) - specific Rab/effector for specific locations v-snares and t-snares tightly associate and assist in the fusion of the membrane

Question 29

What is the recycling pathway?

Answer 29

- the mechanism used to retain resident proteins in the ER - soluble ER-resident proteins contain a 4 amino acid sequence (KDEL, Lys-Asp-Glu-Leu) - A membrane-bound receptor in the cis Golgi binds the ER retention signal and shunts such proteins into special transport vesicles that return the proteins to the ER - this is assisted by the coat protein, COPI