March 30, 2006Pabio552, Lecture 21 For anyone having trouble understanding retrotranslocation, quality control in the ER, and molecular chaperones (from

March 30, 2006 Pabio552, Lecture 2 1

For anyone having trouble understanding retrotranslocation, quality control in the ER, and molecular chaperones (from lecture 1), here are two reviews that should help:

Spiess C, Meyer AS, Reissmann S, Frydman J. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets. Trends Cell Biol. 2004 Nov;14(11):598-604. Review.

Tsai B, Ye Y, Rapoport TA. Retro-translocation of proteins from the endoplasmic

reticulum into the cytosol. Nat Rev Mol Cell Biol. 2002 Apr;3(4):246-55. Review.


Lecture 2: Trafficking from ER to Golgi:Outline:A. Important concepts in trafficking:

1. ER as the gateway to the secretory pathway2. ER translocation is co-translational3. Signals direct translocation across membranes4. Conservation of topology5. Molecular sorting keeps membranes biochemically distinct6. Vesicle targeting involves specific address tags

B. Specific events in vesicular traffic1. Definitions2. Exocytosis3. Lysosomal Sorting4. Endocytosis5. Vesicle fusion (covered in PM lecture)


Trafficking from ER to Golgi: Outline, cont.:

C. Machinery and Mechanisms of Trafficking1. Overview2. Types of coats: Clathrin, Cop I, Cop II3. Proteins that function with coats

D. Experimental Systems1. Cell-free reconstitution of Golgi transport 2. Biochemical analysis of synaptic vesicle membranes3. Genetic dissection of yeast secretion4. EM and Fluorescence microscopy

E. Examples from PathobiologyHow viruses exploit trafficking pathwaysThe lysosomal dogma turned on its head


Trafficking : ER to Golgi to Lysosome A. Important Concepts in Trafficking:

1. The ER as the secretory pathway gateway.Entry into the ER allows trafficking to specific compartments including ER, Golgi, endosomes, lysosomes, plasma membrane and cell exterior.

2. ER translocation is co-translational in higher eukaryotic cells.

In contrast, import into ER of yeast (lower eukaryotes) is post-translational.

In addition, import into nucleus, mitochondria, and peroxisomes is post-translational.

Note that flow in the secretory pathway goes in multiple directions.

The Cell: A Molecular Approach, 3rd Ed (Cooper). ASM Press, 2000


A 2. ER translocation is co-translational


Please review the signal hypothesis and mechanisms of co-translational translocation in the Alberts’ textbook!


Please review the signal hypothesis and mechanisms of co-translational translocation in the Alberts’ textbook!


Trafficking : ER to Golgi to Lysosome A. Important Concepts in Trafficking: 3. Signals direct translocation across membranes.

”N-terminal signal sequences”: the cannonical example of signals that mediate trafficking. Entry into the secretory pathway requires a signal sequence (SS). SS overcome unfavorable energetics in protein transfer across membranes.

Variations in SS: stop-transfer sequences (signal anchor) & internal signals.

At the ER: SS allow interaction of nascent chain with SRP & SRP receptor, leading to cotranslational translocation through the translocon.

Variations in SS directed translocation occurs in bacteria, yeast, eukaryotes, & in different compartments (i.e. co-translational vs. post-translational, translocation into other organelles).

Other types of signals are important in trafficking besides SS - to be covered in upcoming lectures.


Trafficking : ER to Golgi to LysosomeA. Important Concepts in Trafficking:3. Signals direct translocation across membranes.How was the critical role of signal sequences demonstrated?

Translation of a secreted glycosylated protein in a cell-free system +/-tunicamycin (tun), +/- microsomal membranes (mb), +/- protease (pro)added at various times (t):

AAA

+aa,35-S met,GTP,cell extract,(containingribosomes,translationfactors,other factors)

MbPro

t (min)+0 30 0 30

tun+

+_ _ _

++ +

+ _ ++0

____

_

_ _ _ _ _

J. R. Lingappa, Pabio 552, lecture 2-7

microsomes

mRNA transcript

Pro

microsomallumen

cytosol

cytosol

We’ll fill these lanes in during class


Trafficking : ER to Golgi to Lysosome A. Important Concepts in Trafficking: 4. Conservation of topology:

i. Membrane sidedness (lumenal vs. cytoplasmic) is maintained throughout the secretory pathway:

Why? Because there is lateral mobility of lipid & protein in lipid bilayers, but typically no spontaneous flip-flop across bilayers (this is energetically

unfavorable).

cytoplasm

lumen

TGN

extracellular

PM

TVER

lumen



4. Conservation of topology: ii. Lumen = lumen = extracellular space

Why is this? It follows from how the ER evolved in primitive eukaryotes.

Evolution of the Eukaryotic ER



4. Conservation of topology: ii. Lumen = lumen = extracellular space

cytoplasm

lumen

TGN

extracellular

PM

TVER

lumen



4. Conservation of topology:

cytoplasm

lumen

TGN

extracellular

PM

TVER

lumen



4. Conservation of topology:



5. Molecular sorting keeps membranes in each compartment "biochemically distinct" despite continuous vesicular traffic between compartments.

Involves balance of forward and backwards transport.

Types of sorting:A. Selection of specific components during formation of TVB. Segregation of vesicular container from cargo after fusion.C. Retrieval of specific components for retrograde transport.

i.e. proteins bearing KDEL and KKXX sequences bind to specific recycling receptors in the Golgi and are selectively transported back to the ER.

TVER Golgi

TV

= PM TM protein

= Golgi TM protein

= KDEL receptor

= soluble ER protein

= secretory proteinRTVTV = transport vesicle

RTV = retrograde TV



6. Vesicle targeting involves specific address tags that identify donor vesicle and target organelle.

examples: V-SNARES vs. T-SNARES, Rab GTPases that are specific for different organelles or vesicles.



cytoplasm

extracellular

PM

V

TGN

cytoplasm

extracellular

PM

V

TGN

Trafficking : ER to Golgi to LysosomeB. Specific Events in Vesicular Trafficking:1. Definitions:Exocytosis - fusion of vesicles derived from TGN with the PM resulting in insertion of transmembrane proteins into PM or secretion of soluble proteins into extracellular space.

Endocytosis - process by which particles, solutes, membrane proteins (including receptor-ligand complexes) and lipids are taken up by vesicles from the PM. Also used by parasites and bacteria to get into the host cell (to be discussed in Nancy Freitag's upcoming lecture).

Phagocytosis - uptake of pathogens as a defense, clearance of cell debris.

Pinocytosis - uptake of extracellular fluid through endocytosis.


cytoplasm

TV

TGN

extracellular

PM

TV

ER

Trafficking : ER to Golgi to Lysosome B. Specific Events in Vesicular Trafficking2. Exocytosis

a. Events that occur in the ER (review): Translocation, signal cleavage, N-linked core glycosylation

Proper folding by ER resident chaperones Post-translational modifications: trimming core sugars, adding GPI anchors Retention of ER proteins via KDEL sequences

b. Trafficking from ER to Golgi complex: Transport vesicles (TV): bud from mb of one organelle & fuse with mb of next organelleGolgi complex: a series of stacked membranes

where proteins from ER are further processed (i.e. glycosylation, trimming, and CHO addition), & sorted for transport to final destinations: outside cell, PM, or lysosomes

Consists of cis Golgi, Golgi stack (medial and trans), and trans Golgi network (TGN)Distinct polarity of Golgi: entry via the cis face; exit from the trans face.


Trafficking :ER to Golgi to LysosomeB. Specific Events inVesicular Trafficking2. Exocytosis

Direction of traffic:ER to ERGIC to cis Golgito medial Golgito trans Golgito trans Golgi network (TGN)to lysosomes, PM or exterior.

Lingappa, Pabio 552, lecture 2-12



Trafficking : ER to Golgi to Lysosome B. Specific Event, Vesicular Trafficking3. Lysosomal sorting A. Lysosomes: Organelle containing enzymes that degrade

proteins, nucleic acids, CHO and lipids. Delivery from exterior (via receptor-mediated

endocytosis or phagocytosis) or from ER via Golgi.

Lysosomal enzymes: Acid hydrolases (i.e. cathepsin D), Active at pH 5 (pH maintained within lysosomes) Inactive at neutral pH. Protects cell in case of

release into the neutral cytoplasm. Acid pH maintained by ATP-dependent H+ pump in

membrane.

B. Trafficking of lysosomal enzymes:1. 14-sugar N oligo-saccharide core added in the

ER to lysosomal enzymes.2. One mannose and 3 glucoses are removed while

protein is still in the ER.

The Lysosome

J. R. Lingappa, Pabio 552, Lecture 2-14


Trafficking : ER to Golgi to Lysosome

3. Lysosomal sorting B. Trafficking of lysosomal enzymes, cont.:3. Mannose residues on lysosomal enzymes are phosphorylated creating M-6-P residues.

enzyme: N-acetyl-glucosamine (GlcNAc) phosph-transferase, whichrecognizes a specific conformation (signal patch) present only on lys. enzymes.

site: cis Golgi. 4. Another enzyme removes GlcNac leaving M-6-P residue on lysosomal enzyme.



Trafficking : ER to Golgi to

Lysosome

4. Lysosomal sorting, cont. B. Trafficking of lysosomal

enzymes, cont.:

5. The M-6-P residue binds to M-6-P receptors located in trans Golgi. Binding occurs at pH = 6.5 - 7 (pH of Golgi), but not at pH < 6.

6. Clathrin-coated vesicles bud from trans Golgi, become uncoated, & fuse with late endosome.



Trafficking : ER to Golgi to Lysosome 4. Lysosomal sorting, cont. B. Trafficking of lysosomal enzymes,

cont.:

7. Late endosome (LE) pH = 5.5 so lyso enzyme is released from M-6-P receptor. Phosphatase in LE removes phosphate to prevent rebinding.

8. Transport vesicles (TV) transport enzymes to lysosomes

9. Some lysosomal enzymes need to undergo proteolytic cleavage (in lysosome) to become active

10. A different TV recycles M-6-P receptor back to trans-Golgi



Trafficking : ER to Golgi to Lysosome 4. Lysosomal sorting C. Trafficking PM to lysosome:

1. Receptors take up cargo via receptor-mediated endocytosis (next lecture), into clathrin-coated endocytic vesicles which bud into cell from PM and fuse with early endosomes.

2. Early endosomes mature into late endosomes, with recycling of membrane components back to PM.

3. Late endosomes mature into lysosomes, resulting in lowering pH to 5.5, allowing the lysosomal enzyme activation.

Note: M-6-P receptor is also transported to the PM where it binds extracellular phosphorylated lysosomal enzymes that are occasionally secreted. Thus proteins that are accidentally sent to one compartment can be rescued and brought back to another compartment.


Overview of Trafficking to the Lysosome



Trafficking : ER to Golgi to Lysosome

C. Machinery and Mechanisms of Trafficking:

1. Overview of Coats and Adaptors:a. Two functions of coats:

1. Drive budding: deforms planar into curved.2. Select cargo destined for forward transport.

b. Three types of coats:i. clathrinii. COP Iiii. COP II

c. Principles of how coats act:1. Oligomerization of coat proteins into lattices.2. Connection to specific adaptors.3. Additional cellular proteins regulate bud formation

and pinching off.4. Uncoating required for vesicle docking and fusion.5. Coat components are recycled.


J. R. Lingappa, Pabio 552, Lecture 2-21

Clathrin-Coated Vesicles:

Trafficking: ER to Golgi to LysosomeC. Machinery and Mechanisms of Trafficking2. Types of coats:

a. ClathrinActs during uptake of extracellular molecules at PM in endoctyosisActs during lysosomal sorting in the TGNStructure: three-legged trimer of 3 HC and 3 LCOligomerizes to form polyhedral lattice in coated pitUndergoes rearrangement to form curvature that results in buddingAssembly/disassembly regulated by Hsp70 using ATP hydrolysis

Clathrin Structure


Different Coat Proteins Act at Specific Points in the Secretory Pathway


J. R. Lingappa, Pabio 552, Lecture 2-22Trafficking: ER to Golgi to LysosomeC. Machinery and Mechanisms of Trafficking

2. Types of Coats :b. CopI: Made of coatamer subunits.

Mediates retrieval of proteins from Golgi to ER (retrograde transport). COPI vesicles transport ER resident proteins with KKXX or RRXX

signals.Uses GTP binding protein ARF (as does clathrin).Note: The drug Brefeldin A inhibits activation of the ARF protein by

inhibiting nucleotide exchange, and thereby inhibits budding of COPI vesicles.

c. CopII: Mediates forward movement of vesicles from ER to Golgi (anterograde transport).

Regulated by a GTP binding protein Sar1.Budding of COPII is not inhibited by Brefeldin A (which is specific for

Arf).


Trafficking : ER to Golgi to Lysosome C. Cellular Machinery, Coats and Adaptors:3. Proteins that function with clathrin: i. Adaptor proteins promote clathrin assembly, linking clathrin to the membrane and interacting with membrane proteins encoding signals for sorting into CCVs. ii. GTP-binding proteins (include ARF and Sar1) regulate coat protein binding:

Sar1 or ARF bound to GTP recruits coat proteins to vesicle. Coat proteins promote bud formation.After budding occurs, GTP is hydrolyzed to GDP resulting in dissociation of

coat proteins from vesicle.Guanine Exchange Factors (GEFs) exchange GDP and replace with GTP


Model for Dynamin Action

Vesicles in Shibire mutant

Trafficking: ER to Golgi to LysosomeC. Machinery and Mechanisms of Trafficking 3. Proteins that function with coats:

c. Dynamin, a GTPase protein: Localizes to membrane-bud junction to cause vesicle closure (coated pit

becomes vesicle). Purified dynamin can constrict vesicles to form long tubelike structures. Dynamin activity is probably regulated by a kinase-phosphatase cycle. Other proteins (i.e. amphiphysin) implicated dynamin recruitment from cytosol. Temperature-sensitive dynamin mutants in drosophila (shibire) undergo

paralysis due to accumulation of long-neck coated pits and failure to generate coated vesicles in neurosecretory cells.


Trafficking: ER to Golgi to LysosomeC. Machinery

4. Summary



Example ofGenetics UsedFor Studying Trafficking

Trafficking : ER to Golgi to Lysosome D. Experimental systems used to study trafficking: 1. Cell-free reconstitution of Golgi transport (i.e. Rothman & colleagues) Uses cytoplasmic extracts, ER plus Golgi membranes, and de novo synthesis of

radiolabeled proteins off mRNA transcripts to study protein trafficking.Particularly useful for identifying novel cellular machinery.

2. Biochemical analysis of different membranes.

3. Genetic dissection of yeast secretion (i.e. Schekman & colleagues). Sec mutants = yeast mutants defective in various stages of vesicular transport, i.e.

protein secretion, vacuolar transport, or retrieval of ER resident proteins. Isolation of mutants led to molecular cloning of genes.

4. Electron microscopy; Fluorescence microscopy

Molecular Biology of the Cell, 4th edition


+ unlabeled aa,35-S met,GTP, ATPcell extract - ribosomes - factors for translation,translocation, trafficking

microsomes

in vitro transcript

Cell-free systems for studying trafficking

Molecular Biology of the Cell, 4th edition


HIV Env

HBV capsid

ER

GOLGI

HIV virion

PM

ERGIC

NUCLEUS

HIV - Env glycoprotein co-translationally translocatedinto ER as atransmembrane protein,trafficks via secretory path-way to Golgi, where it iscleaved by a furin pro-tease, then moves to PM.Capsid assembles at PMwhere Env is present.

Herpesvirus - Capsidassembly in the nucleus,two models proposed forsubsequent trafficking: de-envelopment at outer nucl.mb. vs. exit within avesicle.

HBV - capsid assembly inthe cytoplasm, buds intoER, exits via secretorypathway.

Coronavirus - assemblesby glycoprotein buddinginto the ERGIC. Helicalcapsid not needed for bud -ding to take place.

How Viruses Exploit the Trafficking Pathways of the Cell during Virion Formation

Coronavirus

Herpes

Trafficking: ER to Golgi to Lysosome E. Examples from Pathobiology:



Additional Reading on Pathogens and Intracellular Trafficking Pathways

Overviews:Knodler, L. A., J. Celli, and B. B. Finlay. Pathogenic Trickery: deception of host cell processes. Nature Rev. Mol. Cell. Bio. 2: 578-588 (2001)(review)

Reprogramming the phagocytic pathway - intracellular pathogens and their vacuoles. Mol. Memb. Biol. 15: 103-121 (1998)

Specific Organisms:Stegmann, T. Membrane fusion mechanisms: The influenza hemagglytinin paradigm and its implications for intracellular fusion. Traffic 1:598 (2000).

Portnoy, D. A., V. Auerbuch, and I. J. Glomski. The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity. J. Cell Bio 158:409-414 (2002) (minireview)

Roy, C. R. and L. G. Tilney. The road less traveled: transport of Legionella to the endoplasmic reticulum. J. Cell Bio 158:415-419 (2002) (minireview)

Russell, D. G., H. C. Mwandumba, and E. E. Rhoades. Mycobacterium and the coat of many lipids. J. Cell Bio 158:421-426 (2002) (minireview)

Andrews, N. W. Lysosomes and the plasma membrane: trypanosomes reveal a secret relationship. J. Cell Bio 158:389-394 (2002) (minireview)

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March 30, 2006Pabio552, Lecture 21 For anyone having trouble understanding retrotranslocation, quality control in the ER, and molecular chaperones (from