HC13 membranes and protein targetting

Question 1

thickness of membrane layers depends on:

Answer 1

level of chain saturation:
- unsaturated: double cis-bonds –> thinner and more fluid. only CIS!!! not trans
- saturated: straight tails –> thicker and more rigid

Question 2

Laurdan fluorescence spectroscopy

Answer 2

used to analyse membrane fluidity. depending on the membrane fluidity more or less water molecules will be present: so a different emision wavelength.

Question 3

membrane phospholipids 4

Answer 3

Head groups can also be charged.
1. phosphatidylethanolamine = not charged
2. phosphatidylserine = - charged
3. phosphatidylcholine = not charged
4. sphingomyelin = not charged. does not contain glycerol as a backbone but contains sphingosine.

Question 4

Phospholipids distrubtion in eukaryotic double membrane

Answer 4

assymmetrically

inside of membrane: phosphatidylserine - charged
outside of membrane: sialic acids (NANA).

Question 5

lipid rafts

Answer 5

cholesterol aids in creating lipid rafts. these rafts have lower lateral mobility.

Question 6

integral membrane proteins.

Answer 6

have hydrophobic regions. often contain helically shaped hydrophobic domains –> to cross hydrophobic membrane.
- isoleucine, leucine, phenylalanine, alanine

Question 7

proteins anchored in membrane via

Answer 7

fatty acids or prenyl groups. no integration of protein into the membrane

Question 8

FRAP

Answer 8

fluorescence recovery after photobleaching
marker proteins are fluorescently labeled –> part of cell is bleached with laser beam –> fluorescence of this patch will be recovered by lateral movement –> determine fluidity/lateral movement speed.

Question 9

Nuclear import/export + signals

Answer 9

do NOT pass a membrane, but a nuclear pore (water channel). this is gated-transport and is dependent on Ran-GTP.
Nuclear export signal (NES)
Nuclear localisation/import signal (NIS/NLS): have internal stretch of 5 positive charged aa (K, R)

Question 10

Ran-GTP/Ran-GDP

Answer 10

make import/export to the nucleus unidirectional. [Ran-GTP] is high in the nucleus, in the cytosol [Ran-GDP] is high.

import: nuclear import receptor binds cargo protein –> receptor-cargo complex enter nucleus via nuclear pore –> Ran-GTP has a high affinfity (low Km) for the receptor –> freeing the cargo protein –> Ran-GTP-receptor through nuclear pore –> Ran-GDP-receptor in cytosol –> let go of each other

export: nuclear export receptor –> receptor binds Ran-GTP in nucleus –> now cargo protein can bind as well –> through nuclear pore to cytosol –> Ran-GTP converted to Ran-GDP –> Ran-GDP dissociates from receptor –> freeing of cargo protein –> receptor goes back into the nucleus.

Question 11

Sec and protein export in gram negative bacteria + powered by?

Answer 11

Protein is synthesised by ribosome –> SecB binds to protein –> keeps it unfolded –> SecB delivers it to SecA+SecYEG –> ATP hydrolysis –> push protein through SecYEG chanel into periplasm (between inner and outer membrane) –> LepB cleaves signal sequence from protein –> protein is folded in periplasm.

Question 12

TAT + powered by?

Answer 12

can move certain already folded proteins accros inner membrane into periplasm. powered by proton motive force.

Question 12

mitochondrial proteins

Answer 12

have N-terminal + charged amphipathic signal sequences.

Question 13

translocation of mito proteins + driven by?

Answer 13

cytosolic hsp70: keeps protein in unfolded state –> ATP hydrolysis to release hsp 70 –> protein binds to TOM complex –> inserted into membrane –> translocated into matrix by TIM23 (driven by membrane potential) –> mito hsp70 binds to polypeptide chain as it becomes exposed to matrix (ATP is required) –> signal peptide is cleaved off by signal peptidase.

• energy drived: needs membrane potential and heat shock proteins.

Question 14

membrane potential over inner mito membrane: arises from?

Answer 14

arises from oxidative phosphorylation:
Pyruvate –> transported over membrane –> into mito –> NADH (reduced compound) –> NADH is taken up by complex 1 –> electrons are taken of –> transported to ubiquinone (electron carrier) –> electrons passed to complex 3 –> electrons are put on cytochrome c (electron carrier) –> passes electrons to complex 4 –> O2 is reduced –> H2O.

During all of these steps: protons are moved into the intermembrane space.
a proton gradient is build up = membrane potential.
H+ flows back into the matrix via ATP synthases.

Question 15

TIM23 dependent sorting to inner membrane of mito (IMM): 2 ways

Answer 15

• through initial import and re-export. Transport of protein into matrix –> N-terminal signal sequence is spliced off –> second signal –> protein is translocated into the IMM
• via stop-transfer signal: very hydrophobic signal. So first a normal signal sequence, then the stop-transfer sequence

Question 16

TIM22 dependent sorting to IMM

Answer 16

through a single internal signal sequence.

Question 17

pHluorin

Answer 17

comes from GFP. its excitation wavelengths depends on pH of the environment. can be used to measure pH of organelles in vivo: making fusion protein: pHluorin with a signal sequence.

pH is mito is higher than cytosolic pH.

Question 18

biogenesis peroxisomes

Answer 18

from the ER. new peroxisomes can originate by fission (splitting in two) or made de novo from vesicles that originate from ER

Question 19

peroxisomes: function

Answer 19

very long fatty acids are degraded/oxidises (Beta-oxidation) in peroxisomes.
Reduced to H2O2 (= toxic) by catalase. (in mito this is NADH).

Question 20

peroxisomal targetting signal

Answer 20

C-terminal: SKL: Serine, Lysine, Leucine
Recognised by soluble import receptors (translocate folded proteins).