!"#$%&'%()*%+*,,%-&,./*%01%02%/*/3452*%3&.26%&4752*,,*1%%

!"#$%&'(")*++*%,*'-&'./%/,*#01#%-2)#3&)/%4'(")*++*% % %5"0)%-2)#3&) %%% %67'2#72'*%%%%%%%%%%%%%%%%%%%%%%%4'(")0/./%%8$+&'&,+"/7 % %,$&7&/9)7$*/0/%%%%%%%%%% %:&2;+*<.*.;= %%%>+")7/?%,'&3/7/%%!@ %%%%%%%% % %.&:01#"3&)?%-&+:0)(%%%%%% %/0)(+*<.*.;=%%%%% %%%A++%%

%%%%%%% % %&-%)*B%,'&7*0)/?%+0,0:/%%C&+(0 %%%%%%%%% % %%/&'3)(?%.&:01#"3&)%%% %%%/0)(+*<.*.;=%%%% %%A++%*2D"'9&7*/%

% % %%&-%)*B%,'&7*0)/%%507& %%%%%%%%%%% % %%*)*'(9%,'&:2#3&)%%%%%%%%%% %%:&2;+*<.*.;=%%%% %%A++%*2D"'9&7*/%%E"#2&+*%%%% %%%%%%%%%%%%%%%%%%%%/7&'"(*%F%$&2/*#+*")0)(%%%%%%%%%/0)(+*<.*.;=%%%% %%A++%*2D"'9&7*/%%G2#+*2/%%%%%% % %HGA%."0)7*)")#*%F%%%%%%%%%%%%%%%%%%:&2;+*<.*.;=%%%% %%A++%*2D"'9&7*/%

%%%%%%%% % %@GA%7'")/#'0,3&)%

C*)*'"+%'2+*I%4'(")*++*/%"'*%(*)*'"7*:%-'&.%7$*%/".*%79,*%&-%&'(")*++*%

%.07&#$&):'0"% 507&#$&):'0"%J.")9K'0;;&)L%

#$+&'&,+"/7/% 8$+&'&,+"/7/%J.")9L%

*):&,+"/.0#%'*3#2+2.%J!@L% *):&,+"/.0#%'*3#2+2.%J!@L%JML%

('&B7$% :0N0/0&)%

)2#+*2/% G2#+*2/%JML%

Cytoplasmic !!!!Nuclear!!!!Mitochondrial!!!Plasma membrane !Secreted !Resident ER and Golgi !Endosomes, Lysosomes!

NO SIGNAL SEQUENCE!

NUCLEAR LOCALIZATION SEQUENCE!

MITOCHONDRIAL SIGNAL SEQUENCE!

ER SIGNAL SEQUENCE!

The endoplasmic reticulum (ER) -- Largest membrane-bound organelle. -- Supply proteins to other organelles in the endomembrane system. -- ~1/3 of total cellular proteins.

The secretory pathway

ER Golgi

cis medial trans ribosome

8M%

PM

Electron Microscopy (EM)

1960’s George Palade Imaging of the endomembrane system

Trafficking of GFP-VSVG

Temperature sensitive vesicular stomatitis virus G protein (VSVG) mutant begins to traffic at permissive temperature.

Temperature shift from 40 oC to 32 oC

Endomembrane system

The endoplasmic reticulum (ER)

Figure 12-8 Molecular Biology of the Cell (© Garland Science 2008)

-- Distinct functions -- One continuous organelle -- Ratio varies across cell types

The rough ER – site for protein synthesis

Pancreatic acinar cell (Similar in other professional secretory cells)

(Lumen)

The smooth ER

Leydig cell from testis

-- Lipid synthesis -- Adapted to specific biological functions -- Detoxification (e.g. cytochrome p450 in liver )

Biochemical assays in cell-free systems

Dounce homogenizer

Figure 12-37b Molecular Biology of the Cell (© Garland Science 2008)

Rough ER

Smooth ER

Cell-free transport assay

Integral Membrane protein

Soluble lumenal protein

Organelle proteins

Cytosol Organelle Lumen

-- These types of proteins are found in all organelles. -- Peripheral membrane proteins are not shown.

Lipid-anchored proteins

Integral membrane protein

Soluble lumenal protein

How are proteins targeted to organelles?

Cytosol Organelle Lumen

Ribosome

Signals must exist to direct proteins:!!1.  Signal sequences for translocation!

2. Sorting or retention signals!

Signal !sequence!

Translocation across the ER membrane !

trans-membrane!(TM) segment!

Soluble!protein!

Membrane!protein!

Cytoplasmic !!!!Nuclear!!!!Mitochondrial!!!Plasma membrane !Secreted !Resident ER and Golgi !Endosomes, Lysosomes!

NO SIGNAL SEQUENCE!

NUCLEAR LOCALIZATION SEQUENCE!

MITOCHONDRIAL SIGNAL SEQUENCE!

ER SIGNAL SEQUENCE!

Table 12-3 Molecular Biology of the Cell (© Garland Science 2008)

Signal sequence directs protein targeting

Each organelle has its own signal sequences for targeting

Figure 12-35a Molecular Biology of the Cell (© Garland Science 2008)

Figure 12-35b Molecular Biology of the Cell (© Garland Science 2008)

8./52%94&021.,02% :;<=:><<?<<;<<;<=@?A?;;;BCDEFF%%%

?4&5,3./02% :G=DHCIJ<<<CJJ;KJB>@DFF%%

K*51(%02-*4(51*%L,&27%94&6.+(M% :<<N;C<C<<;@C;;GIJ;BJ:HFF%%

ER-targeting signal sequences

How do you confirm an ER signal sequence?

Signal !sequence!

Translocation across the ER membrane !

trans-membrane!(TM) segment!

Soluble!protein!

Membrane!protein!

Recognition of signal sequence on the ER

Translocon from bacteria

Figure 12-38 Molecular Biology of the Cell (© Garland Science 2008)

Biogenesis of soluble lumenal proteins

Example: BiP

Figure 12-46 Molecular Biology of the Cell (© Garland Science 2008)

Biogenesis of type I membrane protein

N!

N!

C!

C!

Type I!membrane!protein !

cytosol!

Membrane!

lumen!

N!C!

N!

C!

Other topologies!

_!

Topologies of ER proteins

Soluble !lumenal !protein!

Juxtamembrane residues may influence protein orientation

N!

N!

N!

C!

C!

C!

Membrane proteins have different topologies in ER membrane!

cleaved!signal!sequence!

Type I! Type II!

N!

C!Type III!

cytosol!

ER!

lumen!

-cleavable signal seq.!(7-15 apolar residues)!-transmembrane anchor!stops translocation!

-signal anchor !seq. (18-25 apolar)!-not cleaved!

-reverse signal!anchor!

+! +!

_!_!

+!+!

_! _!

+! +!

+! +!

?!

The topology of a protein is maintained:

819O#1%

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

O$*%&)+9%D)&B)%T/,_V%-".0+9%.*.;*'%0/%@>?RQ=%H*/,07*%;*0)(%";2):")7%0)%7$*%!@?%07%0/%)&7%*//*)3"+%-&'%#*++%%N0";0+079%"):%/**./%7&%+0.07%07/%0)7*'"#3&)/%7&%"%/."++%/*7%&-%/2;/7'"7*/=%%S5,2*T02%"):%+5,4*U+.,02%O$*/*%7B&%+*#3)%#$",*'&)*/%0)7*'"#7%B07$%"):%"//0/7%7$*%-&+:0)(%&-%,'&7*0)/%7$"7%#"''9%.&)&(+2#&/9+"7*:%G<+0)D*:%%(+9#")/=%%

ER chaperones and folding enzymes

Disulfide bonds are common among ER proteins

!"#$%&'(()&*+,-)%((.*#/%"0*%(1!2.3%

Disulfide bonds stabilize proteins and promote folding

Figure 12-50 Molecular Biology of the Cell (© Garland Science 2008)

N-glycosylation site: N-X-S/T

N-glycosylation in the ER

N-glycosylation stabilizes proteins and promotes folding

Properly folded ER proteins can be transported to other organelles in the endomembrane system

Incorrectly folded proteins are retained in the ER

Figure 12-54 Molecular Biology of the Cell (© Garland Science 2008)

Incorrectly folded proteins are eventually degraded in the cytosol

Ron and Walter

ER stress response/unfolded protein response

Vesicular transport

Donor compartment

Acceptor compartment

Vesicles have a size of ~60-90nm

Genetic analysis of the endomembrane system (Randy Schekman, Nobel Prize, 2013)

Budding yeasts

WT Sec12 mutant Sec17 mutant

Genetics: RNA interference

Figure 13-4 Molecular Biology of the Cell (© Garland Science 2008)

Three types of coat proteins for vesicle budding

Figure 13-20 Molecular Biology of the Cell (© Garland Science 2008)

Proteins destined for export are captured by COPII coats

Assembly of COPII coat

Figure 13-13d Molecular Biology of the Cell (© Garland Science 2008)

Dissociation

Lipid transfer protein

Transport vesicles

The Golgi

M%.D+/+N"".+%+++/""'"**%*:DD*:N(7N%N(0:+(`9/%#N(Na)('N%*00"):P()'%%bM%07,/9N"c,%*(*'+0(:""%D)P+7/),*)%7N-:"D'+0(%'7B):,/NPP%:0Dd,aNN%%MeM%*DD7D,90PN%:0(((P7Df%",**0/".N+%7D.D*7"*"9%+(DDN7$"NN%7N,"9-):"P%%MgM%'P"7D:"(3%"(+)N.'00)%*,7"""0"9(%+:D'*(*D)0%+N-:+(((f%:N/++3:)(%%eYM%N-*NN"7)(:%7$+((*:-:P%'N.*$1D+9%DDD7(D:N'D%:)'"NPD+''%*N*D"D"+//%%hVM%P$P"'0*0*/%-9*(*:-/*7%+7'"D-**+)%.:+-'/7.D,%NPDN+*:/:+%DD/:0:*0N+%%hbM%N((/7'0,D0%PP+ND*i)(%D*,/'(0),:%*"N"9(""NP%"(N+/(:P:7%(:+N++$N#,%%YeM%+7+(0*7N((%N.7D+0,/)7%NN,7D)/P0-%/7"/:)P,7N%3DN9*(*',%+7D:)$++(7%%YgM%-:+7(0,,",%'(N,P0*Nf%*0:N)(0+'N%7"*:D(7()D%)D037):P)%'+7,**0*'.%%\YM%N):"*D-"**%:DD+D*'0:7%')*+*/9"9/%+D)P0(:D*D%+((D+//*:D%*7.*D"N**D%%

bVM%0*B+*/$P:"%:0*:a"DDD%*+**0NP,00%/D+9(/"(,,%,7(**:7"*GAV<%%

BiP protein (human)

ER retrieval signal

Retrieval of ER-resident proteins

Golgi!

ER!

Transport!vesicles!

KDEL!Retention/retrieval sequence!

KDEL receptor! low pH!

neutral pH!

The KDEL receptor cycle

Figure 13-4 Molecular Biology of the Cell (© Garland Science 2008)

Three types of coat proteins for vesicle budding

COPI coated vesicles mediate Golgi-to-ER and intra-Golgi transport

W1/0./%(*(4&T06*%%%%%%%%%%%%%%%:522&10651*%II%%%%%%%%%%%%%%%%%%%E.+,*&106*%609)&19)5(51*%%%

Modification of oligosaccharides in the Golgi

Figure 13-32 Molecular Biology of the Cell (© Garland Science 2008)

O-linked glycosylation occurs in the Golgi

or serine

Transport through the Golgi

Exocytic pathway

Donor compartment

Acceptor compartment

Vesicles have a size of ~60-90nm

Figure 13-14 Molecular Biology of the Cell (© Garland Science 2008)

Vesicle docking and fusion

What drives vesicle fusion?

SNAREs (Soluble N-ethylmaleimide – sensitive factor attachment receptors)

(Sollner et. al. Nature 1993) Lab of Jim Rothman

Plasma membrane

Vesicle

The SNARE cycle

trans-SNARE complex

cis-SNARE complex

ATP

NSF

!SNAP

NSF

!SNAP

NSF

ATP !SNAP

NSF

t-SNAREs

v-SNARE

Exocytosis

Figure 13-4 Molecular Biology of the Cell (© Garland Science 2008)

Three types of coat proteins for vesicle budding

COPI coated vesicles mediate Golgi-to-ER and intra-Golgi transport

Two types of exocytosis

Stimuli

Golgi

Extracellular Space

Neurotransmitter release in neurons

Insulin secretion from pancreas

Constitutive exocytosis

Regulated exocytosis

All cell types

Neurotransmitter release in synaptic transmission

[Ca2+] Wormbook

Presynaptic Terminal

Postsynaptic Terminal

H*(52.1%(&T02%

The endocytic pathway - Endocytosis

S,5()402%(401X*,0&2%

?I?Y%01%&2,Z%'&.26%&2%%()*%9,51/5%/*/3452*%

Figure 13-51 Molecular Biology of the Cell (© Garland Science 2008)

Figure 13-53 Molecular Biology of the Cell (© Garland Science 2008)

Figure 13-57 Molecular Biology of the Cell (© Garland Science 2008)

The endocytic pathway - Lysosome

Figure 13-36 Molecular Biology of the Cell (© Garland Science 2008)

Figure 13-43 Molecular Biology of the Cell (© Garland Science 2008)

Transport through the Golgi