Biochem3.protein Targeting.mcv/vcu

Front 1

Advantages of Compartmentalization

Back 1

-Concentration

-Sequestration

-Regulation

Compartmentalization is an energy-intensive process. However, there are several advantages to compartmentalization.

Front 2

List the sites where protein synthesis occurs in mammalian cells

Back 2

1. Mitochondrion

2. Cytosolic ribosomes unbound to membranes

3. Cytosolic ribosomes bound to membranes

Front 3

Secreted and Secretory. The difference.

Back 3

Secreted proteins are a subclass of secretory pathway proteins

Front 4

Protein Targeting (Address) Information

Back 4

-Contained within amino acid sequence of protein

-no “address” = default... synthesized by cytosolic ribosomes, remain “free” in cytosol

-Synthesized on unbound cytosolic ribosomes: nuclear, mitochondrial, peroxisomal

-Secretory pathway proteins --> ER bound cytosolic ribosomes

Front 5

3 Categories for Inter-Compartment Movement

Back 5

1. Vesicular Transport (SNARE, Rab, Coat proteins, etc...)

2. Channels (didn't talk about)

3. Cisternal Progression (only for the golgi)

Front 6

Overview of the Secretory Pathway

Back 6

-secretory pathway proteins can be membrane-associated or soluble

-soluble proteins are localized to the interior of each organelle (lumen)

-start in the ER and move to the golgi

-protein sorting --> trans golgi network (TGN) --> final destinations

Front 7

Targeting of Secretory Pathway Proteins to the ER involving SRP

Back 7

-HYDROPHOBIC signal sequence emerges, binds SRP

-Binding pauses translation, ribosome binds SRP receptor on ER

-Protein enters ER through translocon (channel)

-Signal peptidase removes signal sequence

-Energy for import is derived from hydrolysis of GTP

-Protein import into ER is cotranslational and only unfolded proteins can pass through the translocon.

Front 8

Secretory Membrane Proteins are Retained in the ER Membrane

Back 8

-Most membrane proteins are generated when a second stretch of hydrophobic amino acids “gets stuck” in the ER membrane

-Membrane proteins are classified according to the location of their N-termini (4 types)

Front 9

Protein Topology in the Secretory Pathway. Four types of membrane Proteins

Back 9

Organelle lumen = topologically equivalent to the exoplasmic space (exterior of the cell)

Type I and Type III = N-termini in the lumen

Type II = N-termini in the cytosol

Type IV = more than one membrane spanning domain. Each is a stretch of hydrophobic amino acids

Front 10

Post-translational modification of proteins in the ER

Back 10

-glycosylphosphatidylinositol (GPI) anchor

-disulfide bond formation (PDI)

-folding and oligomerization (chaperones, lectins)

-assembly of N-linked oligosaccharides

-retention / degradation of misfolded proteins

-Note: many of these modifications are co-translational; in other words they occur while the secretory pathway protein is being synthesized and is passing through the translocon channel.

Front 11

GPI Anchored Proteins

Back 11

Hydrophobic Fatty Acid chains

Polar phosphatidylinositol ends (N-terminal)

GPI anchors are important because (1) they anchor proteins in the membrane (2) they promote protein diffusion in the membrane, and (3) they target proteins to the apical plasma membrane of polarized cells.

Front 12

Formation of GPI Anchored Proteins II

Back 12

GPI anchor (modified phosphatidylinositol) is preformed and in ER membrane

Transamidase recognizes amino acids at C-terminus of newly synthesized protein

Transamidase cleaves protein and transfers performed GPI anchor to new C-terminus

Front 13

Formation & Rearrangement of Disulfide Bonds

Back 13

-disulfide bonds form in the lumen; an oxidizing environment

-the cytosol is a reducing environment

-incorrect cysteines can be paired, but disulfide bonds are shuffled until appropriate pairs are formed (disulfide rearrangement or shuffling)

Front 14

Protein Folding and Oligomerization

Guess what? BiP is a chaperone!

Back 14

- Secretory pathway enter the ER unfolded

-Folding occurs in the lumen by molecular chaperones (ex. BiP)

-Chaperones bind hydrophobic regions and prevent premature folding or oligomerization

-Chaperones provide energy by hydrolyzing ATP

-BiP ALSO regulates ER Stress Response (activated by misfolded proteins)

Front 15

There are two major types of glycoproteins:

N-linked glycoproteins
O-linked glycoproteins

Back 15

-O-linked oligosaccharides are assembled in the golgi
-sugars added to serine residues
-added one at time
-relatively simple structures

-N-linked glycoproteins assembly starts in the ER
-modified in the ER and golgi
-attached to asparagine residues
-complicated structures
-three basic types: high-mannose, complex, and hybrid

Front 16

Assembly of N-Linked Glycoproteins

Back 16

-N-linked oligosaccharides are added from a pre-formed precursor

-the precursor is attached to the ER membrane through a lipid called dolichol

-oligosaccharide protein transferase recognizes specific asparagines and transfers the oligosaccharides from dolichol to the newly synthesized N-linked glycoprotein

-after transfer, N-linked oligosaccharides are trimmed in the ER and further processed in the golgi

Front 17

Responses to ER Stress

Back 17

1. Attenuation of new protein synthesis

2. Induction of molecular chaperones (promotes folding)

3. Induction of ERAD (promotes destruction of misfolded proteins)

4. Programmed cell death/ apoptosis (destroys damaged cells)

Front 18

Activation of ER Stress Response

Back 18

-when proteins fold appropriately...
-BiP binds lumenal domains of IRE1, PERK, and ATF6
-this interaction prevents dimerization, activation, and translocation

-when UNFOLDED proteins accumulate...
-BiP is released and binds the unfolded proteins
-release of BiP allows IRE1, PERK, and ATF6 to dimerize, activate, translocate and induce the ER stress response

Front 19

Activation of PERK and IRE1 during ER Stress

Back 19

-During ER stress, BiP is recruited away from PERK and IRE1 to assist with protein folding - remember that BiP is a chaperone

-Loss of BiP allows PERK and IRE1 to homodimerize

-Dimers are enzymatically active and catalyze reactions that result in attenuation of translation (PERK) and induction of genes for protein folding and ERAD (IRE1).

Front 20

ER Associated Degradation (ERAD)

Back 20

-XBP1 pre-mRNA is spliced to its mature form by IRE1, an endonuclease activated by ER stress

-XBP1 protein is a transcription factor that INDUCES proteins required for ERAD and protein folding

-ERAD proceeds through four basic steps: recognition of misfolded protein, retrotranslocation
to cytosol, ubiquitination, and proteosomal degradation

Front 21

Degradation of Misfolded Secretory Pathway Proteins. ERAD.

* ERAD is intended to restore homeostasis, thereby preventing programmed cell death.

Back 21

-Misfolded proteins are retained by molecular chaperones in the ER lumen
-recognized by chaperone that promote retrotranslocation (“dislocation”) to cytosol
-deglycosylated by N-glycanase in the cytosol

-Deglycosylated protein is polyubiquitinated

-Ubiquitinated protein is degraded by proteasome

Front 22

Activation of ATF6 during ER stress

Back 22

-Dissociation of BiP allows ATF6 to translocate to golgi where it is processed, and transcription factor domain is released

-ATF6 transcription factor translocates to nucleus to induce genes encoding ER chaperones

Front 23

General Concepts of Vesicular Transport

Back 23

-vesicles are small, membrane-bound bodies that bud from one organelle and are targeted to and fuse with another

-all non-ER resident secretory pathway proteins undergo vesicular transport. Many ER residents do as well

Front 24

Vesicular Transport

Endogenous proteins (newly synthesized by cell)

Back 24

-ER to golgi (anterograde and retrograde)

-golgi to golgi (retrograde)

-golgi to plasma membrane

-golgi to polarized membrane

-golgi to lysosomes

Front 25

Vesicular Transport

Exogenous proteins (come from outside the cell)

Back 25

-plasma membrane to endosome / lysosome

-phagocytosis
-pinocytosis
-receptor-mediated endocytosis

Front 26

Selective Formation of Transport Vesicles I

Back 26

-coat proteins deposited on membrane of donor organelle

-coat proteins polymerization --> bud --> vesicle formation (GTP required)

-adaptors “connect” membrane proteins in donor organelle with coat proteins; this packages membrane proteins into the bud/ vesicle (selective incorporation)

-two major types of coated vesicles: clathrin and coatomer

Front 27

Targeting of Transport Vesicles

Back 27

-once vesicle has been formed, coat proteins are depolymerized and shed
-requires energy (ATP for clathrin coated vesicles; GTP for coatomer coated vesicles)

-loss of coat proteins exposes targeting proteins in the vesicle (v-SNARES)

-v-SNARES interact with specific t-SNARES in membrane of the target organelle

-this interaction “docks” the transport vesicle at the membrane of the target organelle

-Rab-GTP proteins in transport vesicle bind specific Rab effector proteins in target organelle

-Rab hydrolyses GTP, providing energy to maintain the vesicle-target interaction

Front 28

Fusion of Transport Vesicles with Target Organelles

Back 28

-transport vesicles deliver their cargo to target organelles through membrane fusion event
-SNARE proteins are essential

-NSF and SNAP proteins recylce Rab and SNARE proteins

Front 29

Transport of Secretory Pathway Proteins from ER to Golgi

Back 29

-most proteins leave the ER and are transported to the golgi through vesicular transport

-ER resident proteins are “retrieved” through retrograde transport in COPI vesicles

-this retrieval requires a targeting sequence (“address”) on the ER protein

Front 30

Retrieval of ER Resident Proteins from the Golgi

Back 30

-soluble ER resident proteins have the amino acids K D E L at their C-termini

-these amino acids bind a membrane protein called the KDEL receptor

-the KDEL receptor interacts with adaptors in the COPI complex

-these interactions drive KDEL receptors and their ER resident protein cargo into the bud/transport vesicle that is eventually targeted back to the ER

Front 31

Protein Transport Within the Golgi

Back 31

-anterograde transport within the golgi by “cisternal progression”

-each stack (cisterna) moves “forward”, taking the position of the stack ahead of it

-this moves both golgi resident and non-resident proteins

-resident golgi proteins are retrieved to their appropriate stacks through retrograde transport (COPI)

Front 32

Destinations of Proteins that Pass Through the Golgi

Back 32

-golgi resident proteins

-continuous secretion / plasma membrane (default)

-regulated secretion / secretory vesicle (requires signal)

-lysosomes (requires signal)

-polarized secretion / expression (requires signal)

Front 33

Secretory pathway proteins that are sorted in the TGN:

1. Regulated Secretion

2. Polarized Secretion

3. Endosome/ lysosome

Back 33

1 & 2. Regulated and Polarized secretion, involve coated vesicles, adaptors, SNAREs, Rab, etc

-endosome/ lysosome targeting involves clathrin coated vesicles

-clathrin coated vesicles are also involved in the import of exogenous proteins (receptor-mediated endocytosis)

Front 34

Regulated Secretion

Back 34

-typically signaling molecule (ligand) binds cell surface receptor

-binding increases cytosolic calcium

-Calcium signal induces transport of secretory vesicles to plasma membrane

Front 35

Polarized Cells I

Back 35

-epithelial cells have specialized regions of the plasma membrane that have specific functions and therefore specific proteins. Thus, they are “polarized cells”.

-GPI anchored proteins are exclusively transported to apical membrane; apparently GPI anchor is an “address”.

Front 36

Polarized Secretion II

Back 36

-Direct sorting: apical and basolateral proteins are packaged into separate vesicles in the TGN

-Indirect sorting: apical and basolateral proteins are packaged into the same vesicles in the TGN

-basolateral and apical proteins are endocytosed and delivered to endosomes

Front 37

Lysosomal Protein Sorting

Back 37

-Soluble lysosomal proteins have N-linked oligosaccharides that are modified to generate the “address” that targets them to the lysosome

-they receive a mannose-6-phospate (M6P) tag (address) in the cis golgi

-M6P binds M6P receptor in the TGN; M6P receptor also interacts with adaptors that bind clathrin coat proteins

-once transport vesicle is formed, clathrin coat depolymerizes generating an “early endosome”

-early endosome is targeted to late endosome through SNAREs, Rab proteins

Front 38

Receptor-Mediated Endocytosis

example: LDL

Back 38

-LDL receptor is located in “clathrin coated pits” in the plasma membrane

-ligand binding triggers receptor binding to adaptor (AP2) that then binds clathrin

-this interaction induces clathrin polymerization and formation of coated vesicle

-clathrin depolymerizes, generating early endosome that is targeted to late endosome

-in late endosome, ligand (LDL) targets to lysosome; receptor is targeted back to plasma membrane