Lecture 11: Golgi and Lysosomes-Function and Trafficking

Question 1

Golgi complex

Answer 1

• Ordered series of flatten membrane stacks called cisternae
• Usually consist of 4-6 cisterna
• Each of the Golgi cisterna have unique protein (and lipid) composition
• Stacks are often connected by tubules to form a continuous organelle
• Located in the perinuclear region (around the nucleus) close to the centrisome
• Localization is maintained by microtubules

Question 2

Model of the Golgi Complex

Answer 2

Question 3

Which stains can be used to indicate the different golgi compartments?

Answer 3

Trans face- Nucleoside diphosphate

Cis face- Osmium

TGN- Acid phosphatase

Question 4

___ proteins cause coating with COPII in the golgi

Answer 4

Sec proteins

Question 5

The ____ complex begins to build a coat

Answer 5

Sec23/Sec24

Question 6

The ___ complex finishes the coat

Answer 6

Sec13/31

Question 7

Coat proteins also help sequester ____

Answer 7

cargo receptors

Question 9

In what 3 ways are cargo selected for inclusion in vesicles?

Answer 9

• Export signals
• Retention
• Bulk flow

Question 10

Export Signals

Answer 10

Proteins to be transported have a export signal that allows selective packaging into transport vesicles

Question 11

Retention

Answer 11

• Proteins have a mechanism to be excluded form transport vesicles
• Chaperone binding
• Kin Recognition

Question 12

Bulk flow

Answer 12

– Some proteins get packaged simply because they are in the right place at the right time

Question 13

_____ also participates in decision about entry into COPII transport vesicles and ER exit

Answer 13

• The ER quality control apparatus
• Most of the time this is a good thing to prevent inappropriately folded protein or subunits of unassembled complexes from reaching the cell surface
  
  • Large amounts (up 90%) of the T-cell receptor, acetylcholine receptor are degraded and never reach the cell surface

Question 15

In what case is the quality control machinery is too good?

Answer 15

– Cystic Fibrosis Transmembrane Regulator (CFTR)

Question 16

ER-derived transport vesicles fuse to form ____

Answer 16

vesicular tubular clusters (VTC)

Question 17

VTC’s form by ____ membrane fusion

Answer 17

homotypic

Question 18

What happens when ER resident proteins get transported out of the ER?

Answer 18

They are actively retrieved!

– Soluble residents

• KDEL sequence and receptor

– Membrane bound residents

• KKXX motif

Question 19

How does the KDEL receptor know when to bind and when to release KDEL-containing proteins?

Answer 19

• The interaction between the KDEL tetrapeptide and the KDEL receptor is pH sensitive.
  
  • The relatively acidic pH of the Golgi compartment permits the association of the KDEL tetrapeptide with the KDEL receptor
  • The more neutral pH of the ER allows release (and prevents binding)

Question 20

The KDEL receptor binds to escaped KDEL protein in the ____

Answer 20

cis Golgi

Question 21

ER-Golgi anterograde and retrograde traffic

Answer 21

Question 22

_____ function to anchor and localize transport gesicles

Answer 22

Golgins

Question 23

How do golgins do anchor and localize vesicles?

Answer 23

Through a rab interaction domain and a cytoskeletal interactor domain

Question 24

What are the two models of intra-golgi transport?

Answer 24

1. Vesicle transport model
2. Cisternal maturation model

Question 25

Vesicle transport model

Answer 25

Question 26

Cisternal Maturation Model

Answer 26

Question 27

Biochemical functions of the Golgi

Answer 27

• Glycosylation – Modification of existing Nlinked sugars – O-linked glycosylation – Production of proteoglycans * Protein sorting * Sulfation – Specific tyrosine residues in proteins

Question 28

Biochemical function of the cis golgi network

Answer 28

* phopsphorylization of oligosaccharides on lysosomal proteins

Question 29

biochemical functions of cis cisterna

Answer 29

* removal of Man

Question 30

Biochemical function of medial cisterna

Answer 30

* removal of Man * addition of GlcNAc

Question 31

Biochemical functions of trans cisterna

Answer 31

* addition of Gal * addition of NANA

Question 32

Biochemical functions of trans golgi network

Answer 32

* sulfation of tyrosines and carbohydrates

Question 33

What is the purpose of protein glycosylation?

Answer 33

• Aid in protein folding – ER quality control • Surface coating – Since animal cells do not have a cell wall, the extended surface coating by sugars provides come degree of protection from the extracellular environment • Specific interactions – Some sugars provide interaction surfaces for cell-cell recognition • Extracellular Matrix

Question 34

What's the signal for glycosylation?

Answer 34

--Asn-X-Ser/Thr--

Question 35

Two main classes of N-linked glycans?

Answer 35

1. Complex oligosaccharides (Gal and NANA) 2. High-Mannose oligosaccharide (Man)

Question 36

Order of sugar modifications?

Answer 36

1. Glucoses removed by glucoxidases in ER 2. Some Mannoses removed by mannosidases in both the ER and Golgi (to become High-mannose when has 6-Man) 3. GlcNAc added by N-acetylglucosamine transferase I and Man further removed 4. Step 3 repeats until 3 Man are connected each to a GlcNAc, Gal, and NANA

Question 37

Sugar modification diagram

Answer 37

Question 38

In Proteoglycan production (O-linked glycan) sugars are added....

Answer 38

in the golgi and as repeating disaccharides

Question 39

O-linked glycans are very ____ while N-linked are very\_\_\_\_

Answer 39

O= straight N= highly branched

Question 40

Lysosomes

Answer 40

• Primary degradative organelle – Degrades proteins, lipids and other cellular constituents taken in by endocytosis, phagocytosis, and autophagy • Other functions – Stores osmolytes – Stores nutrients as well as waste products • Heterogenous in size and function

Question 41

What's the hallmark of autophagy?

Answer 41

double membrane

Question 42

Stages of autophagy?

Answer 42

Question 43

• Most lysosomal proteins (including the hydrolases) leave _____ and are diverted to the lysosome

Answer 43

the secretory pathway

Question 44

How are lysosomal proteins segregated from other secretory proteins?

Answer 44

They contain a specific modification on their oligosaccharides - a phosphate group on the 6-position of a terminal mannose (M6P)

Question 45

How is M6P added?

Answer 45

* Lysosomal proteins contain a “signal patch” that is seen by the enzyme N-acetylglucosamine phosphotransferase (GlcNAc) * This enzyme binds both the high mannosecontaining lysosomal protein to be modified and UDP-GlcNAc and catalyzes the transfer of GlcNAc phosphate to a terminal mannose residue * The GlcNAc sugar is removed by a glycosidase leaving the mannose-6-phosphate

Question 46

Inclusion-Cell (I-cell) disease is caused by a mutation in \_\_\_\_\_

Answer 46

GlcNAc phosphotransferase

Question 47

Where does the Mannose-6 phosphate receptor (M6PR) reside?

Answer 47

Trans-golgi network

Question 48

M6PR binding is ___ sensitive

Answer 48

pH, dissociates in acidic conditions of early endosome