Lecture 15: Glucose Transport

Question 1

Name the three modes of transport for Na+, which is the symporter and which is the antiporter? What drives the Na/glucose symporter?

Answer 1

Na/k ATPase, Na/glucose symporter and glucose transporter (GLUT) antiporter. The na/k atpase creating high conc of extracellular Na+

Question 2

Describe the properties of GLUTs.

Answer 2

1. faster transport than diffusion as don’t need to move through the hydrophobic bilayer
2. are specific to a single type of molecule
3. reversible transport dependent on conc. gradient

Question 3

What is the KM for glucose transport and what does this mean?

Answer 3

1.5mM so at this conc half the glucose transporters have bound glucose and transport is 50% of the max. rate.

Question 4

How many TM domains do human GLUTs have? What are all family members? Where are the N and C terminal? how many classes, which TM are important for substrate binding?

Answer 4

12, glycosylated at different sites, cytosol, 3, TM7, 11, 12.

Question 5

Which is the best understood and where is it found?

Answer 5

GLUT1, at the PM of most mammalian cells especially on the erythrocyte PM.

Question 6

Why have GLUTs been studied in erythrocyte cells lots?

Answer 6

because they have no nucleus or organelles and easy to purify.

Question 7

How are GLUT1 and 4 alike?

Answer 7

Sugar moiety found in both, similar substrate binding residues, similarity in intracellular loops and domains.

Question 8

What is XylE?

Answer 8

A d-xylose transporter found in E.coli

Question 9

What is the architecture of XylE?

Answer 9

12 TM formed of 2 x 6 helical domains with cytoplasmic N and C terminals.

Question 10

Where is the sugar binding site of XylE? What is its substrate?

Answer 10

toward the C terminal and the substrate, D-cylose can access it from either side of the protein.

Question 11

What residues form the proposed binding site of XylE?

Answer 11

Q282, Q283, N288 from TM7 and N317 from TM8 and N415 from TM11.

Question 12

What is the N domain of XylE responsible for?

Answer 12

regulating the conf changes required for transport

Question 13

What does the intracellular domain of XylE do?

Answer 13

Block the xylose from being released.

Question 14

Compare the structures of XylE and GLUT1-4.

Answer 14

intracellular helices are conserved in GLUT1-4 and the residues that mediate inter domain interactions are conserved between XylE and GLUT1-4. There is a lot of conservation in the transmembrane domains.

Question 15

How does D-xylose coordinate to XylE?

Answer 15

it is hydrogen bonded to the C domain by polar and aromatic residues. The aromatic residue, Tyr298 may prevent prevent D-xylose leaving to the extracellular side.

Question 16

Which mutation in XylE has no effect?

Answer 16

N325

Question 17

What can inhibit the transport of D-xylose and why?

Answer 17

D-glucose because they have similar chemical structures

Question 18

What has more prominent function in binding D-glucose? How?

Answer 18

Gln168 as it forms 3 H bonds with it

Question 19

What is similar between D-glucose recognition and GLUT1-4?

Answer 19

all residues apart from 2 that mediate D-glucose recognition are invariant in GLUT1-4

Question 20

What does the latest GLUT1 model have?

Answer 20

4 alpha helices on the intracellular side

Question 21

What should be very similar and why?

Answer 21

Recognition of D-glucose by GLUT1-4 as should be nearly identical to that observed in D-glucose bound to XylE as the vast majority of AAs involved in substrate binding are invariant between Cyle and GLUT1-4.

Question 22

Mutations at which amino acids are predicted to impair conformational flexibility?

Answer 22

Gly73 and Gly130

Question 23

When was the structure of GLUT1 predicted and from what?

Answer 23

2012-13 from bacterial

Question 24

What happens in the mammalian brain? In what tissues is this the opposite?

Answer 24

They have constitutively high glucose requirement so GLUTs1-3 and constitutively targeted to the cell surface. Muscle and adipose tissue

Question 25

How to erythrocyte cells ensure more glucose will keep entering the cell?

Answer 25

Glucose is phosphorylated to glucose -6-phosphate which cannot leave the cell back through a GLUT and means intracellular glucose levels are low so glucose keeps coming in down the conc gradient.

Question 26

Where is GLUT4 predominantly expressed??

Answer 26

Adoposytes and muscle cells.

Question 27

Where inside the cell is GLUT4 mainly distributed?

Answer 27

endosomes, trans-golgi network and endosomal sorting intermediate structures and GLUT4 storage vesicles (GSVs)

Question 28

What happens to GFP-Glut4 when trigger cells with insulin?

Answer 28

get aggregation at the PM from

Question 29

What is the ICH domain of GLUT1?

Answer 29

The intracellular helix bundle

Question 30

What does GLUT4 translocate to the PM in response to?

Answer 30

Inslulin

Question 31

What proportion of the insulin stimulated glucose uptake occurs in skeletal muscle and adipocytes?

Answer 31

90 and 10%

Question 32

What are GSVs and what are they formed from?

Answer 32

GLUT specialised vesicles. Endosomes or TGN.

Question 33

What do GSVs consistantly do but what limits their binding?

Answer 33

Sample the PM but are limited by lack of docking sites or tethering proteins.

Question 34

How does ARF6 drive vesicle formation?

Answer 34

it is on the donor membrane and it rectuite adapter proteins which interact with clathrin and GSV resident proteins

Question 35

What does the insulin receptor initiate and What protein creates targetting sites for the GLUT4 GSVs?

Answer 35

TC10 GTP bound

Question 36

What does the insulin receptor initiate the pathway of? What is the consequence?

Answer 36

PI3K signalling cascade which recruits PISK which converts PIP2 to PIP3 which recruite PDK1 and AKT. Activation of AKT by PDK1 promotes GSV exocytosis

Question 37

What is defective in diabetes 2?

Answer 37

Translocation of GLUT4 to PM

Question 38

What is glucose stored as in adiposytes?

Answer 38

Fat