Professor Ugalde lecture 8

Question 1

On what side of the membrane are phoshpholipids synthesized?

Answer 1

cytosolic side of the ER membrane

Question 2

Where are fatty acids attached and how are head groups added in phoshpholipid synthesis?

Answer 2

fatty acid (acyls) are attached to coenzyme A in chemically reactive states
-Glycerol phosphate, head group added in sequence by enzymes

Question 3

Where are phospholipds and cholesterol synthesized?

Answer 3

cytosolic side of the ER membrane

Question 4

What protein in the ER membrane flips lipids rapidly in lipid synthesis?

Answer 4

Scramblase flips lipids randomly and it is an ATP independent function

Question 5

How are lipids transported after they have been synthesized?

Answer 5

transported through secretory pathway by vesicles, and then later they are flipped to correct orientation

Question 6

What protein maintains membrane assymetry at the plasma membrane?
What is the role of this protein, is it energy dependent, is it specific/what for?

Answer 6

Flippase protein maintains membrane assymetry by flipping from outer membrane to cytosol, it is ATP dependent, directional, and lipid specific

Question 7

What are the 3 ways lipids are transported

Answer 7

VCO
V-by vesicles between organelles and secretory pathway
C-by carrier proteins through cytosol
O-through contact sites between organelles

Question 8

In between what organellese can components of the membrane be exchanged by contact?

Answer 8

in the ER and mitchondria

Question 9

what type of information does the sequence of the protein determine and what does the amino acid sequence determine?

Answer 9

structure, function, and localization
-amino acid sequence determines where the protein should be anchored

Question 10

what does localization of membrane proteins require, and what does the structure involve contact with?

Answer 10

-localization of membrane proteins requires protein based targeting mechanims
structure of membrane proteins involves contacts with lipids

Question 11

Where/how are integral membrane proteins tightly anchored?

Answer 11

they are tightly anchored by hydrophobic interactions with the interior of the lipid bilayer

Question 12

What are the different types of integral membrane proteins?

Answer 12

1. 1 or more transmembrane alpha helices
2. transmembrane beta-barrel
   3.amphpathic alpha helix in one face of the membrane (one polar and hydrophobic side

Question 13

What are lipid anchored proteins, and what does their strength depend on?

Answer 13

-they are proteins which are covalently linked or one or more lipids/fatty acid groups
-the strength of the anchor depends on number and type of lipid

Question 14

What peripheral membrane proteins attached by?

Answer 14

non covalent interactions

Question 15

what are the 2 types of peripheral membrane protein/what are they interacting with?

Answer 15

-they are interacting strongly with integral membrane proteins
-they also have weak interactions with lipid head groups

Question 16

where do transmembrane proteins function, what do they act as?

Answer 16

they function in both cellular compartments (lumen/exterior and cytosol)
-they act as cell surface receptors and transporters

Question 17

what are the characteristics of lipid-anchored proteins?

Answer 17

-they function in one side of the membrane
-they take part in intracellular signaling

Question 18

what is the most common form of attachment?

Answer 18

transmembrane alpha helices

Question 19

where do amino acid side chains point in alpa helices

Answer 19

they point outwards

Question 20

in transmembrane helices why are the side chains hydrophobic, and what maintains the structure of the helix?

Answer 20

-side chains are hydrophobic to interact with lipids
-internal hydrogen bonds maintain structure of helix

Question 21

How do transmembrane helices differ than soluble (cytosolic proteins)?

Answer 21

TM helices are longer and more hydrophobic, consisting of 18-24 amino acids long since they have to be long enough to be inserted in the PM.

Question 22

how do you predict if transmembrane helices are present in a protein?

Answer 22

can be predicted from the hydrophobicity of primary sequence

Question 23

how does the length of TM helices relate to the membrane?

Answer 23

length of TM helices matches the width of membrane

Question 24

what do longer TM helices partition into?

Answer 24

they partition into thicker microdomains, or insert at an angle in thinner membranes

Question 25

What is the advantage of just have one TM helix

Answer 25

the protein can rotate easily

Question 26

What can multiple TM helices fold together into?

Answer 26

they can fold together into functional structures embedded in the membrane

Question 27

What does the interior of the TM helices not contain

Answer 27

the interior is free of lipids

Question 28

where does the polar group of TM helices face?

Answer 28

they face the cytosol making hydrophillic interactions

Question 29

Why is the interior of the TM membrane polar?

Answer 29

to allow polar groups to get transported through (creating a hydrophillic environment, common for ions)

Question 30

how are transmembrane barrels formed, and what determined what can be transported through them?

Answer 30

beta sheets wrap into a cylinder, how wide the beta sheet is determines what can be transported

Question 31

where do the side chains point out in TM beta barrels, and what holds the strands together on the inside?

Answer 31

side chains point out into the lipid bilayer -and on the inside hydrogen bonds hold strands together

Question 32

how is transmembrane protein orientation determined?

Answer 32

during its insertion into the membrane, there is some directionality

Question 33

in the secretory pathway how are lumenal/extracellular domains modified differently from cytosolic domains, and what do they stabilize?

Answer 33

-there are disulfide bonds between cysteins -oligosaccharides (glycosylation) -they also stabilize protein structure

Question 34

what mods are only found in the cytosolic domains

Answer 34

phosphorlyation, ubiquitination, acetylation, methylation

Question 35

do membrane proteins flip?

Answer 35

no they do not, unlike lipids

Question 36

what controls the movement of ions and polar molecules across membranes, and what does this mechanism depend on?

Answer 36

-controlled by proteins, also allows for regulated flow of ions -mechanism depends on the concentration gradient of the solute

Question 37

differences between a channel and pump/ transporter

Answer 37

answer in pic

Question 38

How are the TM helices formed water filled channel controlled? What characterisitcs do the channel proteins have?

Answer 38

opening and closing is controlled by cytosolic domains or subunits, and some TM helices must be polar on one side -they are also selective for ions

Question 39

How are voltage gated sodium channels controlled

Answer 39

-pore is controlled by voltage sensor, and other domains

Question 40

what does the channel pore contain?

Answer 40

it contains a selectivity filter for specific ions (ex. K+)

Question 41

What lines the pore of channel proteins?

Answer 41

carbonyls from peptide backbone line the pore

Question 42

what can ions do in the channel protein with water?

Answer 42

ions normally bind with water, and ions with the right size can exchange water for carbonyls (partial dipoles)

Question 43

What happens when the wrong size ion tries to bind to channel protein?

Answer 43

wrong size ion cannot bind carbonyls and is rejected

Question 44

what are examples of some proteins that use ATP hydrolysis to transport substrates

Answer 44

Na+-K+ pump maintains ion gradient across PM

Question 45

Whhat are ATP-binding Cassette (ABC) transporters used to transport?

Answer 45

small molecules like cholesterol, toxins, and phospholipid, flippasses

Question 46

What domains does a multidrug resistance transporter have? What do they do?

Answer 46

ABC transporter with 2 symmetrical ATPase domains -they pump toxins out of cancer cells- resistance to chemotherapy

Question 47

What are the 3 steps of a multidrug resistance transporter?

Answer 47

1. when there is no nucleotide-inward open, high affinity for substrate 2. ATP bound-closed 3. ATP hydrolysis: outwards open, low affinity for substrate (releases toxin out)

Question 48

How can cytsolic proteins be linked in lipid anchored proteins?

Answer 48

cytosolic proteins can be covalently linked to acyl (fatty acid) or prenyl chains)

Question 49

what does the single lipid chain provide in lipid anchored proteins?

Answer 49

it provides transient interactions with the membrane

Question 50

how many lipids are needed for a strong membrane anchor? where do specific enzymes attach?

Answer 50

two or more lipid chains needed for strong membrane anchor -specifci enzymes attach lipid to N-terminus or Cys side chain

Question 51

What can Cys-S-acylation regulate?

Answer 51

-they can be used to regulate interactions with membranes and they are reversible (thioester)

Question 52

What is the difference betweem acylations and prenylation

Answer 52

N-termminal acylations are permanent -prenylation has special branched lipids which are permanentl attached to Cys (thioether)

Question 53

what is the sulfhydryl bond in terms of reactivity?

Answer 53

-it is chemically reactive

Question 54

What does disulfide bond formation do in terms of structure/bond?

Answer 54

-disulfide bond formation (in the lumen of the ER they are covalent and stabilize structure)

Question 55

WHat is a lipid modifcation

Answer 55

thoester,thioether--->acylation

Question 56

What are ubioquition E1 and E2 doing?

Answer 56

-ubiqiotin E1 activating and E2 conjugating enzymes (thioester)

Question 56

What are ubioquition E1 and E2 doing?

Answer 56

-ubiqiotin E1 activating and E2 conjugating enzymes (thioester)

Question 57

what does the prenylation motif do, and what is its sequence?

Answer 57

-it anchors proteins to the membrane -it has Caax-COO- (a=alkyl side chain, x=any) -aax sequence is cleaved off, Cys is prenylated

Question 58

How do proteins become GPI anchored?

Answer 58

Some proteins have special TM helix removed and become covalently linked to glysosyl-phosphatidyl-inositol (GPI) anchor -it has a strong membrane attachment

Question 59

What does GPI protein provide, and what faces the lumen/exterior

Answer 59

-provides mobility to protein in the lumen, more than TM helices -sugar usually faces lumen or exterior (not cytosol)

Question 60

Where are GPI proteins attached?

Answer 60

only on lumenal/extracellular side;attached at ER, function at exterior of PM (extracellular matrix (neuronal receptors..)

Question 61

What happens to the structure of an integral membrane protein in the absence of a membrane? what about lipid anchored and peripheral proteins?

Answer 61

the integral membrane protein will have a diff 3D structure bc its hydrophobic AA will not interact with fatty acids and instead will collapse in the interior of the protein. -Lipid anchored and peripheral proteins will have the same structure since they are just anchored