14. golgi and vesicular traffic

Question 1

Golgi apparatus

Answer 1

• golgi conissts of a stack of membrane-bounded cisternae located b/w the endoplasmic reticulum and the cell surface - resembles a stake of hollow pancakes
• named after camillo golgi (1898)
• primary function - to modify and sort proteins for export to other organelles
• golgi is cells shipping department

Question 2

golgi structure

Answer 2

bottom to top (vesicle traffic in 2 directions, transitional elements, no ribosomes)
Cis golgi network
cis cisternae
medial cisternae
trans cisternae
trans-golgi network (network of tubules , vesicles) sorting area
to: secretory vesicles (lysosomes, cell membrane)

Question 3

each compartment (cis,medial,trans) has unique enzymes:

Answer 3

functional differentation of the golgi complex can be studied with the electron microscope with specific techniques that detect different enzymes:

• cis region is where sugars and proteins are phosphorylated
• the medial (middle) region have enzymes that remove carbohydrates that were added in the ER, and then add new carbohydrates
• the trans region is the area where the lysosomes are sorted. therefore, it is rich in acid phosphatase, an enzyme found in lysosome

Question 4

golgi function

Answer 4

stepwise glycosylation of proteins (adding sugars):
the making of glycoproteins:
-N-linked glycoproteins
-Continued processing of N-linked sugars (from ER)
-O-linked glycoproteins

Question 5

N-linked glycoproteins

Answer 5

sugars attached at the N of the asparagine amino acid (also occurs in the ER)

Question 6

Continued processing of N-linked sugars (from ER)

Answer 6

• substrate is a sugar-nucleotide (activated sugar), which is transported into the Golgi from cytoplasm
• Series of glycosyltransferases, which act in a rigidly determined sequence

Question 7

O-linked glycoproteins

Answer 7

-sugars attached to the O of serine of threonine amino acids

only occurs in golgi

Question 8

why glycosylate proteins ?

Answer 8

• makes proteins more hydrophilic
• allows them to bind to the extracellular matrix
• reduces their susceptibility to proteases(partly b/c carbohydrates are more rigid and thereby can provide physical barriers to the core of the protein)
• provides another level of structural complexity to proteins, which can:
• give the protein a new function
• provide more specificity in its interactions with other proteins

Question 9

where do we see an abundance of glycosylated proteins ?

Answer 9

on the cell’s surface, where cell to cell interactions occur

Question 10

What did John hodgson say about glycoproteins?

Answer 10

almost without exception, whenever two or more living cells interact in a specific way, cell surface carbohydrates will be involved

Question 11

Golgi is a transient structure which means its …

Answer 11

constantly changing

Question 12

the following contents all move through the layers of cisternae:

Answer 12

• membranes including proteins, phosphoglycerides, other membrane lipids
• materials within vesicles including proteins being processed and the enzymes doing the processing

Question 13

two models of transport through the golgi:

Answer 13

• cisternal maturation model

- vesicular transport model

Question 14

cisternal maturation model

Answer 14

each cisterna is physically moved up the stack and changes composition.
evidence: there are some materials, including large molecules that are never seen in the vesicles

Question 15

vesicular transport model

Answer 15

• materials in the interior and in the membrane are brought up to next cisternae by vesicle transport
  evidence: vesicles were found to move both forward and backward (anterograde and retrograde), so they could maintain enzyme differences

Question 16

movement of vesicles

Answer 16

• not random of mediated by diffusion, as people once though
• vesicles travel along microtubules
• anchored to microtubules by various proteins, including one called dynactin (only with dynein,not kinesin; kinesin attaches with help of other adaptor proteins)
• vesicles propelled along microtubules by motor proteins, such as dynein and kinesin

Question 17

is the golgi a permanent organelle?

Answer 17

no
the golgi breaks up and disappears at the onset of mitosis
by telophase of mitosis, the golgi reappears
we are still uncertain how it is recreated
growing microtubules may help collect and align small vesicles which later fuse together and the plus end

Question 18

vesicular traffic

Answer 18

• vesicles 60-100nm diameter, deliver soluble and membrane-bound molecules by fusion of membranes
• vesicles are covered by proteins

Question 19

three functions of the vesicle’s integral and peripheral proteins:

Answer 19

1. vesicle formation/budding
2. selection of vesicle contents by receptor system
3. recognition and fusing to target membrane

Question 20

three types of vesicles

Answer 20

1. COP II-coated vesicles
2. COP I-coated vesicles
3. Clathrin-coated vesicles

Question 21

COP II-coated vesicles

Answer 21

forward movement from ER and b/w cisternae

-anterograde movement

Question 22

COP I-coated vesicles

Answer 22

retrograde movement from TGN to golgi to RER

Question 23

Clathrin-coated vesicles

Answer 23

TGN to lysosomes and endocytosis

Question 24

vesicles selectively transfer the following materials through the cell:

Answer 24

• contents w/in vesicles
• bulk flow, non-specific contents eg. water
• specific materials (proteins, carbohydrates, lipids) concentrated within
• specific membrane proteins

Question 25

Some requirements of vesicular transport

Answer 25

• selection of contents
• receptor mediated
• production of vesicles
• by protein coat
• movement of the vesicles
• movement, tethering/docking, membrane fusion

Question 26

selection of vesicle contents

Answer 26

-this requires receptors to select and carry specific contents

Question 27

receptors are:

Answer 27

proteins, usually integral membrane proteins
bind other molecules (ligands) with very high specificity and very high affinity
-specificity and affinity give receptors the power to concentrate specific molecules

Question 28

selection of vesicle contents by receptors

Answer 28

the coating of proteins inside a vesicle is the same as the inside of the target compartment and to the outside of the cell
ex. page 17

Question 29

function of protein coats on vesicle exterior

Answer 29

• interact with the cytoplasmic tails of receptors
• ER export signals
• interact with other trans membrane proteins
• put mechanical force on membrane to form vesicles

Question 30

three steps of protein coats on vesicle exterior

Answer 30

1. when coat proteins assemble at the membrane, they force the lipid bilayer to ben
2. coat proteins help gather the receptors that will carry cargo inside the vesicle
3. shortly after the vesicle pinches off, the coat falls off and the vesicle is ready to travel to destination

Question 31

COP II coated vesicles

page 21

Answer 31

coat contains several separate proteins
mechanisms of action:
1.receptors bind selected proteins within the lumen of the RER
* proteins destined for secretion
*ER resident proteins such as BIP are left behind
2. Sar1, a GTP binding protein, if active promotes assembly of the coat
3.coat proteins bind to cytosol side of specific receptors in RER membrane
4. coated bud forms, leaves the RER
*taking along receptor/cargo complexes
*also some membrane proteins (eg.. golgi glycosyltransferases)
*also membrane proteins involved in docking/fusion: destination tags-Rab and SNARES -not shown)
5.Sar1 hydrolyzes the GTP to GDP becomes inactive
6. disassembly of the coat
*things in the lumen of the vesicle and in the membrane stay there
7. the vesicle is now able to fuse with the cis golgi

Question 32

COP 1-coated vesicles

Answer 32

-like COP II but the associated GTP binding protein is called ARF1
-retrograde movement
*trans to cis Golgi
*cis Golgi back to ERGIC, ER
COP I vesicles retrieve proteins-even though the COP II coated vesicles have used receptors to take desired protein from the ER and to allow the ER to retain most of its enzymes, there is some loss, due to bulk flow (ie a little unwanted extra protein gets dragged into the budding vesicle), and it needs to be sent back to the ER- these proteins will have retrieval signals to allow them to be returned

Question 33

COP I coated vesicles have specific retrieval signals

Answer 33

• recognized by the receptors in the COP I membranes as they form:
• soluble proteins (eg protein disulfide isomerase and BiP) have a retrieval signal sequence Lys-Asp-Glu-Leu or “KDEL” that is bound to a KDEL receptor
• membrane proteins (Eg KDEL receptor) have a signal KKXX (Lys-Lys-?-?) at the protein C-terminal
• the KKXX retrieval signal binds to COP I proteins
• return to ER