Secretory Pathways- Lectures 33-35

Question 1

What organelles are involved in the secretory pathway?

Answer 1

Endoplasmic Reticulum (protein folding and quality control)
Golgi Apparatus (sorting)
Lysosome (recycling cellular materials that can be engulfed by membrane invagination)
Lipid Vesicles (transportation)

Question 2

What are the two types of ER?

Answer 2

rough (ribosomal) and smooth

Question 3

How are new proteins targeted in the ER?

Answer 3

N-terminal signal peptide (8-20 residues and enriched hydrophobic amino acids) targets newly made proteins
internal targeting sequences may not be cleaved after import so signal peptide binds to Signal Recognition Particle (SRP) - a ribonucleoprotein complex that attaches to newly synthesized proteins while they are still being translated

Question 4

What happens when SRP binds a peptide?

Answer 4

translation is stalled and the complex of translating ribosome and SRP bind to the ER membrane (via SRP receptor complex) –> translation is resumed when positioned next to the translocon (adjacent to the SRP receptor site) where it can continue translation through the aqueous channel and into the lumen of the ER (or channel can open sideways into the plane of the membrane to create membrane spanning domains)

Question 5

The ER is an ____ environment, where as the cytosol is a ____ environment.

Answer 5

oxidizing

reducing

Question 6

Why is it important that the ER is an oxidizing environment?

Answer 6

because it helps to facilitate folding of proteins that must exist outside the cell (also an oxidizing environment)

Question 7

Proteins entering the ER are glycosylated on _____ via a ____ residue carbohydrate structure that includes _____.

Answer 7

asparagine (N-linked glycosylation)
14
3 residues of glucose, 9 residues of mannosse, and 2 residues of N-acetyl glucoamine (GlcNac)

Question 8

When are proteins entering the ER glycosylated on asparagine?

Answer 8

when it is part of the sequence Asn-X-Ser or Asn-X-Thr

Question 9

Describe the sequence of events that is required for protein to be released from the ER.

Answer 9

the terminal two glucose residues are trimmed from the core glycosyl unit (resulting in monoglucosylate form) –> binding to calnexin (part of quality control apparatus) until fully folded –>
IF properly folded, last glucose is cleaved and protein can be released from the ER and onto the next stage of the pathway
IF not properly folded, glucose is added back until it is properly folded

Question 10

What are the two kinds of membrane proteins?

Answer 10

Type I (N-terminus in the lumen of the ER)
Type II (C-terminus in the lumen of the ER)
Topology Complex (multiple membrane-spanning domains)

Question 11

_____ are lectins- proteins that bind carbohydrates.

Answer 11

Calnexin and Calrecticulin

Question 12

Describe the ER quality control pathway.

Answer 12

capacity of chaperone apparatus is exceeded by unfolded protein –>

1. activation of signaling pathway unfolded protein response (UPR) –> expression of genes that encode ER-specific molecular chaperones and components of ubiquitin/proteasome pathway
2. ER-associated degradation (ERAD)- luminal and membrane proteins are retrotranslocated from the ER to the cytosol for degradation by proteasome

Question 13

Once proteins fold properly in the ER, they begin their journey via _____ to the _____.

Answer 13

lipid vesicles

the Golgi apparatus

Question 14

Describe the structure of the Golgi apparatus.

Answer 14

stack of flat, membranous disks with directionality (cis to trans end movement of proteins)

Question 15

What occurs in the Golgi stacks?

Answer 15

post-transcriptional modifications (eg. trimming of carbohydrates, phosphorylation, and sulfation)

Question 16

What occurs in the trans Golgi network?

Answer 16

proteins sorted from each other and packaged into transport vesicles

Question 17

What does the operation of the retrieval pathway require?

Answer 17

KDEL receptors that recognize a KDEL sequence at the C-terminus of resident ER proteins that are accidentally sent to the Golgi complex in vesicles

Question 18

What are the key characteristics of a lysosome?

Answer 18

built for recycling/digesting anything
full of acid hydrolases
consistently have a pH of 5 (maintained by proton pumps)

Question 19

Describe the travel of acid-hydrolases to the lysosome.

Answer 19

1. acid hydrolases in cis golgi have carbohydrates that are recognized by phototransferase –> phosphorylation manose @ 6 position
2. protein in membrane of trans golgi (pH of 6) that has a receptor allowing for specific vesicles to emerge that just have this receptor and phosphorylated lysosomal protein
3. vesicles fuse with endosome (pH 5.5) where the pH difference causes reduction in affinity of mono 6 phosphate receptor for enzyme –> proteins in the endosome can then transfer the two individually to their respective destinations

Question 20

Provide examples of lysosomal storage diseases.

Answer 20

Gaucher’s Disease (glucocerebrosidase mutation resulting in improper processing of sphingolipids- can be treated with transfusions of mono6phosphate which can enter due to the few receptors that accidentally end up in the plasma membrane during the return from lysosome)
Fabry’s Disease (mutation in alpha-galactosidase A- can be treated with DGJ replacement therapy)

Question 21

What is the difference between constitutively secreted proteins and regulated secretion?

Answer 21

Constitutive (eg. extra-cellular matrix proteins)- travel directly to the plasma membrane and fuse
Regulated (eg. hormones, enzymes, neurotransmitters, and milk proteins)- require a specific signal before they can fuse with the plasma membrane

Question 22

What is the difference between proteins targeted to the basolateral membrane and the apical membrane?

Answer 22

basolateral- have distinct target sequences
apical- get there by several different mechanisms (including specific targeting sequences and glycosylphosphatidyl inositol anchors that are attached in the ER) via lipid rafts

Question 23

What are the main types of endocytosis?

Answer 23

phagocytosis/ pinocytosis (differentiated by size)
receptor mediated endocytosis
autophagy

Question 24

Describe phagocytosis.

Answer 24

macrophages (professional scavenging cells) invaginate very large areas of the plasma membrane to encompass pathogenic bacteria
normally for particles up to about 0.5u in diameter

Question 25

Describe pinocytosis.

Answer 25

how most cells invaginate and form small vesicles to help recycle membranes and transport materials into cells

Question 26

Describe receptor mediated endocytosis.

Answer 26

absorption of nutrients and other biologically active materials into cells via receptors

Question 27

Provide an example pathway of how receptor mediated endocytosis works.

Answer 27

Iron is taken into cell when bound to transferrin that can bind transferrin receptors, which then follows a vesicle pathway to the endosome where the receptor-transferrin complex dissociate from the iron so that they can be sorted separately (the receptor-transferrin vesicle normally goes back to the membrane it came from)

Question 28

Describe autophagy.

Answer 28

mechanism of delivering intracellular components to the lysosome for destruction and recycling via the formation of double membrane structures that engulf organelles or protein aggregates and then fuse with lysosome

Question 29

What are the three types of coat proteins?

Answer 29

clathrin, COPI, and COPII

Question 30

What do COPII proteins do?

Answer 30

coat vesicles that transport from the ER to the Golgi

Question 31

What do clathrin proteins do?

Answer 31

coat vesicles transport from trans-golgi network to the plasma membrane as well as from endosomes to lysosomes

Question 32

What to COPI proteins do?

Answer 32

coat vesicles transporting in the retrograde (ER retrieval pathway) as well as the golgi transport between layers

Question 33

Describe the flow of vesicle formation.

Answer 33

cargo molecules bind to receptors bound to adaptin which are in complex with a coat protein –> conformational change directed by receptor bulges membrane until it is spherical and dynamin can pinch off the membrane –> vesicle uncoats –> naked transport vesicle can go to target site

Question 34

How do naked transport vesicles bind target membranes?

Answer 34

Rabs bind to different vesicles and proff-read by interactign with specific tethering proteins on target membranes –> t-SNARE on target membrane interacts with v-SNARE on vesicle, and their affinity for each other binds the two tighter and tighter until they membranes fuse –> SNARE proteins are disassembled after fusion via specific chaperone proteins

Question 35

What proteins maintain the specificity of vesicles for their target membranes?

Answer 35

Rabs (small GTP binding proteins)

SNAREs