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Lecture unit 2: Biosynthesis and Lecture unit 2: Biosynthesis and vectorial targeting of secretory d b i and membrane proteins Part 1
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Unit 2

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Jonathan Shao

Bio 102 unit 2
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Page 1: Unit 2

Lecture unit 2: Biosynthesis andLecture�unit�2:���Biosynthesis�and�vectorial targeting�of�secretory

d b iand�membrane�proteins��Part�1

Page 2: Unit 2

Overview�of�the�biosynthesis�and�sorting�problem:��Questions�to be addressed will include:to�be�addressed�will�include:

1.��How�were�the�sites�of�synthesis�and�subsequent�processing�of�secretory and�membrane�proteins�experimentally�id tifi d?identified?

2.��What�is�the�evidence�that�proteins�are�usually�inserted�into�the�lumen�of�the�ER�coͲtranslationally?

3.��What�is�the�molecular�basis�for�targeting�mRNAs�to�the�ER�versus�free�cytosolic polysomes?

4. What determines the precise transͲbilayer4.��What�determines�the�precise�trans bilayertopography�of�integral�membrane�proteins?

Page 3: Unit 2

Overview�of�the�biosynthesis�and�sorting�problem:��Questions�y g pto�be�addressed�will�include:

5.��How�does�the�ER�translation�machinery�distinguish�between a secretory or resident luminal compartment proteinbetween�a�secretory or�resident�luminal�compartment�protein�from�an�integral�membrane�protein?

6.��What�types�of�coͲand�postranslation processing�of��d b i h d hsecretory and�membrane�proteins�occur,�where�do�these�occur,�

and�what�are�the�functions�for�such�processing�events?7.��Once�synthesized�in�the�ER,�how�is�a�given�protein�

targeted�to�its�proper�destination?

Page 4: Unit 2

R h d l i i l di d bi h i fRough�endoplasmic�reticulum�mediated�biosynthesis�of�secretory,�plasma�membrane�integral�proteins,�resident�organelle�lumenaland�membrane�proteins

1. In�addition�to�plasma�membrane�integral�proteins�and�secretory proteins�what�organelle�membrane�proteins�and�

id t l i l t i th i d /i RERresident�luminal�proteins�are�synthesized�on/in�RER.��

2. What�organelle�proteins�are�not�synthesized/inserted�at�the�RER?

Page 5: Unit 2
Page 6: Unit 2
Page 7: Unit 2

The�secretory/�exocytic(and endocytic) pathways(and�endocytic)�pathways

Traffic�from�compartment�to�compartment�is�mediated�by�carrier�

l h b d ff f h dvesicles�that�bud�off�from�the�donor�compartment�and�fuse�with�the�recipient�compartment.�These�vesicles�p pcarry�both�membrane�and�luminal�cargoes.

Page 8: Unit 2

Diagram�and�rotary�shadowed�image�of�polyribosomes

Page 9: Unit 2

A�common�pool�of�ribosomal�subunits�assemble�both�cytosolicand�ER�bound�polyribosomes.

Page 10: Unit 2

Rough endoplasmic reticulum; thin section TEM ER LumenRough�endoplasmic�reticulum;�thin�section�TEM.��ER�Lumen

Page 11: Unit 2

En�face�view�of�polyribosomes on�cytoplasmic surface�of�the�RER.��Note�spiral�arrays�of�ribosomes of�varied�lengthlength�

Page 12: Unit 2

What�TEM�method�was�used�to�generate�this�3�D�view�of�the�ER?�

Page 13: Unit 2

Experimental dissection of the secretory pathwayExperimental�dissection�of�the�secretory pathway

Pulse�chase�analysis�of�the�synthesis�and�sorting�of�t t i b th i ti i llsecretory proteins�by�the�exocrine�pancreatic�acinar cell.��

Acinar cells��synthesize�and�store�in�secretory granules�a�large�variety�of�digestive�enzymes.��After�a�meal,�a�peptide�hormone�stimulates�release�(via�exocytosis)�of�these�enzymes�into�ducts�connected�to�the�small�intestine.�

Page 14: Unit 2

LMLM�histologicallystained�section�of�a��pancreatic�acinus.

What�are�the�darkly�stained�granules?granules?�

Pancreatic�duct�for delivery offor�delivery�of�digestive�enzymes�to�small�intestine

Page 15: Unit 2

The�arrow�tracks�the�temporal�and�spacial route�taken�by�newly�synthesized�secretory proteins.Why�was�the�

pancreatic�acinar cell�a�good�system�to investigateto�investigate�the��secretorypathway?

Page 16: Unit 2
Page 17: Unit 2

Thin sectionThin�section�TEM�of�the�RER,�Golgi�region�of�a�pancreatic�acinar cell.

Page 18: Unit 2

Cartoon�depiction�of�a�pulse�chase�experiment.���There�is�continuous��flow (e.g. of newly synthesized protein) from compartment AͲ>D. Toflow�(e.g.�of�newly�synthesized�protein)�from�compartment�A >D.��To�track�temporal�and�spacial aspects�of�flow,�a�label�(e.g.�labeled�amino�acid)�is�added�to�the�system�for�a�brief�time�(The�pulse)�and�its�fate�in�space at various times after removing the label is trackedspace��at�various�times�after�removing�the�label�is�tracked.

RER

Golgi

Condensing vacuole;Condensing�vacuole;Secretory granule

Page 19: Unit 2

Pulse�chase�analysis�of�secretory protein�synthesis�in�the�pancreatic�acinar cell

1. Cut�pancreas�into�thin�slices.��Why�is�thin�important?

2 Incubate slices in medium containing 3H (or 14C)Ͳleucine for ~ 3 min2. Incubate�slices�in�medium�containing� H�(or� C) leucine.�for� �3�min.�THE�PULSE

3. Remove�slices�from�pulse�medium�and�place�into�new�medium�with�3. Remove slices from pulse medium and place into new medium withexcess�“cold”�leucine

4. At�various�times�remove�slices�and�either�a)�process�for�thin�section�EM�or�b)�cell�fractionation/organelle�isolation.��THE�CHASE.

Page 20: Unit 2

5a Cut thin sections cover with film emulsion and develop5a.��Cut�thin�sections,�cover�with�film�emulsion�and�develop�after�2Ͳ4�weeks�to�visualize�exposed�silver�grains�(grids�are�stained�with�uranyl acetate�and�lead�after�developing�film)or

5b.��Place�organelle�fractions�in�scintillation�counter�and�quantify levels of radioactivityquantify�levels�of�radioactivity.�

6.��Quantify�numbers�of�silver�grains�over�RER,�Golgi�secrotorygranules as a function of time.granules��as�a�function�of�time.

Page 21: Unit 2

Diagram�of�an�EM�section�preparation�for�AR�analysis

Page 22: Unit 2

0’ h 45’ h0’�chase

20’�chase

45’�chase

120’�chase

Page 23: Unit 2

Cartoon�of�pulse�chase�results.�Silver�grains�are�shown�in�red

Page 24: Unit 2

pulse�was�0Ͳ3�min

Condensing�vacuoles

Page 25: Unit 2

Initial�steps�in�the�coͲtranslational�targeting�and�translocation�of�a�nascent�polypeptide�chain�into�ER�lumen;�from�Pollard�and�Earnshaw.

Page 26: Unit 2

Figure 13.6 Lodish et al. Cotranslationaltranslocation.

Page 27: Unit 2

1.��Translation�begins�in�cytosol;�signal�sequence�emerges�from�ribosome.��2.�Signal�Recognition�Particle�(SRP)�binds�to�signal�sequence�and�arrests�translation.��3.��SRP�binds�to�ER�SRP�receptor.��4.��SRP�releases�and�SRP�receptor�p ppromotes�insertion�of�NͲterminal�signal�sequence�(as�hairpin�loop)��into�translocon pore�which�is�locked�into�open�state�by�ribosome�binding.

Page 28: Unit 2

5.��ER�membrane�bound�signal�peptidase�cleaves�off�NͲterminal�signal�sequence.��6.��Cotranslationalg qinsertion�of�the�growing�chain�continues.��7�and�8.��Chain�termination�results�in�ribosome�release�from�ER discharge of completed chain into ER lumen andER,�discharge�of�completed�chain�into�ER�lumen�and�closure�of�the�translocon pore.�

Page 29: Unit 2

E i l h i i f RER di d h i dExperimental�characterization�of�RER�mediated�synthesis�and�insertion�of�secretory proteins:

Use�of�isolated�rough�microsomes isolated�from�secretory cell�for�in�vitro�protein�synthesis�assays:

ͲͲDemonstration that proteins are inserted into RERDemonstration�that�proteins�are�inserted�into�RER�lumen�coͲnot�post�translationally

ͲDetermination�that�the�Signal�sequence�is�generally�l d l i llcleaved��coͲtranslationally.

ͲIdentification�and�characterization�of�the�signal�recognition�particle.

ͲCharacterization�of�an�aqueous�pore�in�RER�membrane�through�which�the�nascent�polypeptide�is�cotranslationallyinsertedinserted.

Page 30: Unit 2

Tool�kit�needed�for��these�studies

1. Isolated�rough�ER�vesicles�(rough�microsomes)2 Polyribosomes isolated from rough microsomes2. Polyribosomes isolated�from�rough�microsomes3. “Stripped”�microsomes derived�from�rough�

microscomes by�removing�polyribosomes from�rough�i b fmicrosome membrane�surface.

4. In�vitro�protein�synthesis�system.

Page 31: Unit 2

Preparation�of�microsome and

microsome pellet�is�composed ofmicrosome and�

cytosolic fractions�from�tissue�homogenate by

composed�of�vesiculated elements�of�plasma�membrane,�RER SER endosomehomogenate�by�

differential�sedimentation.��

RER,�SER�,�endosomeand�Golgi.

Page 32: Unit 2

Rough and smooth microsomes are then separated by sucrose densityRough�and�smooth�microsomes are�then�separated�by�sucrose�density�gradient�sedimentation.��

Page 33: Unit 2

Thin section TEM of RER and a pellet of RER derived roughThin�section�TEM�of�RER�and�a�pellet�of�RERͲderived�rough�microsomes.

RER�rough�microsomes

Page 34: Unit 2

Thin sectionThin�section�TEM�of�rough�microsomesdand�ͲͲͲ

polysomesdetached�from�ro ghrough�microsomesusing�non�ionic�detergent.

Page 35: Unit 2

Experimental�dissection�of�b d tmembrane�and�secretory

protein�biosynthesis:

evidence for rapid insertionͲͲevidence�for�rapid�insertion�into�RER�lumen:

e g pulse label tissue slicee.g.�pulse�label�tissue�slice,�then�immediately�isolate�microsomes;��add�protease,�

di ti f lno�digestion�of�newly�synthesized�protein.

Jonathan Shao
If control doesnt work, need to select other protease
Page 36: Unit 2

Isolation of “stripped”—polysome free microsomes from roughIsolation�of� stripped —polysome free�microsomes from�rough�microsomes:

1. Extract�rough�microsomes with�high�salt�and�puromycin.��What�is�the�purpose�of�the�puromycin?

2.��Collect�stripped�microsomes by�high�speed�sedimentation.

Page 37: Unit 2

Evidence�for�coͲtranslational�insertion:

Experiment�1:��

a) Isolate�rough�microsomes from�cells/tissue�making�predominantly�single�protein

b) isolate�stripped�microsomes (using�salt�and�puramycin)�and� c)�detached�) pp ( g p y ) )polysomes (by�detergent�treatment)

c) Perform�in�vitro�translation�assays�using�35SͲmethionine�to�identify�synthesized products The following assays are performed:synthesized�products.�The�following�assays�are�performed:

Stripped�microsomesmicrosomes

b

R h Detached

c

Rough�microsomes

Detached�polysomes

Page 38: Unit 2

Assay�1:��Rough�microsomes (RM)�+�protein�synthesis�cocktail�(PSC)

Assay�2:��Detached�polysomes (DP)�+�PSC

Assay�3:��DP+�SM+�PSC

After�30�minutes�split�each�asay mix�into�2�aliquots;�treat�one�with�protease.��Analyze�the�6�aliquots�from�the�3�assays�by�SDS�PAGE�autoradiography�(AR):��Result:

RM DP SM+DP

NoteProcessed Note�higher�Mr

Processed�and�protected

Processed�and�

Ͳ + Ͳ + Ͳ +� protease

protected

Jonathan Shao
Text
Page 39: Unit 2

Experiment�2:��Does�protease�protection�(luminal�insertion)occur�coͲ or�postͲtranslationally?

Assay 1: DP+SM+ PSC (same as above; control)Assay�1:��DP SM �PSC�(same�as�above;�control)

Assay�2:�a)��DP�+�PSC�for�30�minutes;

b) add protein synthesis inhibitorb)�add�protein�synthesis�inhibitor

c)�add�SM.��

d) split into two aliquots and treat one with protease: SDS PAGE ARd)�split�into�two�aliquots�and�treat�one�with�protease:�SDS�PAGE�AR�Conclusion:��completed�proteins�cannot�insert�post�translationally

Assay�1 Assay�2

NoteNote�higher�Mr

Ͳ + Ͳ +�protease

Page 40: Unit 2

Evidence�that�removal�of�the�signal�sequence�is�cotranslational1. Isolate�stripped�polysomes (as�before�from�cells�making�mostly�a�single�

t t i )secretory protein)2. Use�as�substrate�for�in�vitro�protein�synthesis�under�conditions�that�

only�nascent�chains�can�be�completed,�but�new�chain�initiation�is�dprevented.

3.��Remove�aliquots�at�various�times�and�analyze�the�finished�products�by�SDS�PAGE�AR:

5’ 3’

Green�portion�of�completed�chain�is�radioactive5 3

5’ 3’

Step�1 Step�23���2�������1

1����2�������3

Nascent chains completed chainsNascent�chains����������������completed�chains

Page 41: Unit 2

State�of�polysomes isolated�from�rough�microsomesif��processing�were�CoͲ or�Post�translation.

5’3’ Cotranslational

removal�of�signal�sequence

5’3’

Post�translational�removal�of�signal�sequencesequence

Page 42: Unit 2

Evidence�for�coͲtranslational�removal�of�NͲterminal�signal�sequence.�

h

g

First�nascent�chains�completed�are already

Last�nascent�chains�completed�were�not�processed.�i.e.�were�are�already�

processed;�i.e.�signal�sequence�

d i

pnot�long�enough�for�signal�peptidase�to�cleave off signalwas�removed�prior�

to�isolation�of�rough�microsomes

cleave�off�signal�sequence.

1�����4����10��15���20��30��min.

Page 43: Unit 2

Evidence�for�coͲtranslational�processing�of�the�NͲterminal�signal�sequence�from�secretory proteins:�Blobel and�D bb t i JCB 1975Dobberstein:�JCB:�1975

This�AR�shows�the�synthetic�products�from�polysomes�added�to�in�vitro�protein�synthesis�assay�under�conditions�that�only�allows�completion of attachedcompletion�of�attached�nascent�chains.��Note�that�earliest�time�points�the�signal�sequence�has�been�removed.��The�preͲform�of�Ig�light�chain�appears�only�in�later�time�points.�

Diagram�what�this�gel�would�look�like�if�removal of signalremoval�of�signal�sequence�were�postͲtranslational.

Page 44: Unit 2
Page 45: Unit 2

Examples�of�some�signal�peptide/sequences:

+�basic�aa

~10Ͳ30�hydrophobic�aa

Page 46: Unit 2

How�would�you�experimentally�demonstrate�y p ythat�the�signal�sequence�is�both�necessary�and�sufficient�for�targeting�secretory proteins�to�the�lumen of the RER?lumen�of�the�RER?

Jonathan Shao
Text
Page 47: Unit 2
Page 48: Unit 2

1.��Translation�begins�in�cytosol;�signal�sequence�emerges�from�ribosome.��2.�Signal�Recognition�Particle�(SRP)�binds�to�signal�sequence�and�arrests�translation.��3.��SRP�binds�to�ER�SRP�receptor.��4.��SRP�releases�and�SRP�receptor�p ppromotes�insertion�of�NͲterminal�signal�sequence�(as�hairpin�loop)��into�translocon pore�which�is�locked�into�open�state�by�ribosome�binding.

Jonathan Shao
Co translational=happening while protein is made
Page 49: Unit 2

SRP�has�several�distinct�functions�mediated�by�different�SRP�protein subunits: a) signal sequence binding; b) translationprotein�subunits:��a)�signal�sequence�binding;�b)�translation�arrestͲ c)�SRP�receptor�binding�and�d)�GTP�binding/hydrolysis

Page 50: Unit 2
Page 51: Unit 2

Experimental�characterization�of�SRP�translation�arrest�properties:

Assay�synthesis�(using�in�vitro�protein�synthesis�cocktail)��of��detached�polysomes (or�reconstituted�polysomes using�mRNA�encoding secretory protein of choice; e.g. preͲprolactin ) in theencoding�secretory protein�of�choice;�e.g.�pre prolactin )�in�the�absence�and�presence�of�SRP:

Ͳ +�SRP

Th 70 tid

PreͲprolactin

The�70�aa peptide�represents�the�~�40�aa within�the�

~�70�aa peptide

ribosome�pore�and�~30�aa of�SS�sequence protrudingsequence�protruding�from�large�ribosomal�subunit:�

Page 52: Unit 2

Evidence�for�an�aqueous�channel�for�chain�translocation:��release�of�nascent�chain�from�ribosome�with�puromycin creates�a�ion�permeantp y pchannel�that�closes�upon�release�of�ribosome�from�membrane.��

Page 53: Unit 2

puromycin�mimicks�an�p yincoming�aaͲtRNA�but�cannot�form�a�peptide�bond�and�thus�causes�release of the nascentrelease�of�the�nascent�chain�from�ribosome,�opening�aqueous�channel�in�ER�membrane

Page 54: Unit 2
Page 55: Unit 2

A�lipid�bilayer (called�a�black�lipid�film)�is�formed�across�a�small�hole�in�a�plate�and�then�RMs�are�added�to�one�side�of�the�plate;�the�only�‘route:” for current/ion flow is through the lipid filmroute: �for�current/ion�flow�is�through�the�lipid�film.

Page 56: Unit 2

How�would�you�demonstrate that thedemonstrate�that�the�effect�of�puromycin is�specific;�e.g.�maybe�it�is�altering�bilayerpermeability.

Page 57: Unit 2
Page 58: Unit 2

The�ribosome�is�critical�for�maintaining�the�open�state.��Salt removal of ribosomes results in channel closure.Salt�removal�of�ribosomes results�in�channel�closure.��

Page 59: Unit 2
Page 60: Unit 2
Page 61: Unit 2

M d l f t l ti l i ti fModels�for�co�translational�insertion�of�secretory and�membrane�proteins�through�th RER bthe�RER�membrane

Page 62: Unit 2

Insertion�and�processing�of�a�secretory protein

Page 63: Unit 2

Insertion�of�Type�1�single�spanning�transmembrane protein�with�a�cleavable�signal�sequence.

Page 64: Unit 2

Insertion�of�Type�2�single�spanning�transmembrane protein

Page 65: Unit 2

Internal�start�transfer�and�stop�transfer�sequences�for�insertion�of�multiͲmembrane�spanners.

Page 66: Unit 2

I t l i l i ti f T 1 i lInternal�signal�sequence�insertion�of�Type�1�single�spanning�transmembrane protein;�no�signal�sequence�cleavage.

Page 67: Unit 2

Start�and�stop�transfer�sequences�in�a�multi�spanning�membrane�protein;�these�sequences�become�the�spanning�p q p gdomains�in�the�completed�protein

Jonathan Shao
Only start requires SRP
Page 68: Unit 2

Determination�of�integral�membrane�protein�topography�during�biosynthesis�on�the�RER:

Key determinants:Key�determinants:

1.��NͲterminal�“signal�sequence”�consisting�of�a�hydrophobic�core��of�i id G ll t i f l bamino�acids��Generally�contains�consensus�sequence�for�cleavage�by�

signal�peptidase�CͲterminal�to�the�hydrophobic�core.��This�forms�a�loop�which�promotes�transfer�of��growing�chain�CͲterminal�to�the�signal�sequence.���diagrammed��here�as�NͲSSSSS

2 Internal hydrophobic “stop transfer” sequence segment—2.��Internal�hydrophobic� stop�transfer �sequence�segmentconsists�of�a�hydrophobic�,�alpha�helical�segment�that�arrests�chain�transfer��and�becomes�a�transmembrane segment�in�the�completed�

t i Th d d b ith N t i l i lprotein.��These�are�preceded�by�either�an�NͲterminal��signal�sequence�or�an��internal�start�transfer�sequence.��diagrammed�here�as��XXXXX

Page 69: Unit 2

Determination�of�integral�membrane�protein�topography�during�biosynthesis�on�the�RER:

3.��Internal�start�transfer�sequence:��Like�the�NͲterminal�signal�sequence,�consists�of�a�hydrophobic�segment�flanked�on�either�the�NͲ or�CͲterminal�side�by�charged�(generally�+)�amino�acids.��This�will�promote�chain�insertion�of��protein�chain�segments�either�CͲterminal�(+ charge on NͲterminal side of hydrophobic core) or NͲterminal (+(+�charge�on�N terminal�side�of��hydrophobic�core)�or�N terminal�(+�charge�on�CͲterminal�side�of�hydrophobic�core)�to�the�start�transfer�sequence.����+TTTTT��or�TTTTT+

Page 70: Unit 2

Sequence�key�for�protein�insertion�demo

Protein 1: N-SSSSS-----------------------------------------CProtein 2: N-SSSSS-----------------XXXXX--------------------CProtein 3: ---------------------TTTTT+------------------------CProtein 4: ----------------------+TTTTT----------------------CProtein 5: ----------------+TTTTT-----------XXXXX--------CProtein 6: N-SSSSS---------XXXXX---------------+TTTTT----------C

Jonathan Shao