BIOLOGY 2B03 Lecture Notes - Lecture 11: Secretion, Golgi Apparatus, Exocytosis
3 May 2018
School
Department
Course
Professor
Golgi&complex&
Proteins&travel&as&cargo&via&vesicles&from&the&ER&to&the&Golgi&complex&
•
The&Golgi&complex&is&comprised&of&a&series&of&elongated&flat&sacs&called&
cisternae
•
Vesicles&transport&proteins&to&the&portion&of&the&cisternae&known&as&the&cis-
cisternae
•
Vesicles&leave&the&Golgi&complex&from&the&trans-cisternae,&there&are&two&
secretory&pathways:&
Constitutive&secretory&pathway
Used&by&proteins&that&are&released&immediately&after&protein&
synthesis&and&transport&
-
The&vesicles&can&fuse&with&the&cell& membrane&releasing&the&
protein&outside&the&cell&in&a&process&known&as&exocytosis&
-
I.
The®ulated&secretory&pathway&
Used&by&proteins&that&are&kept&in&the&cell&until&a&signal&triggers&
release&
-
The&secretory&vesicles&held&in&the&cell&are&called&secretory&granules&
-
II.
•
Other&proteins&carried&away&from&the&Golgi&trans-cisternae&in&vesicles&form&the&
lysosome&by&fusing&with&vesicles&at&the&cell& membrane&called&endosomes,&they&
capture&and&transport¯omolecules&from&outside&the&cell&
•
Instead&of&antibodies,&fluorescently-labeled&wheat&germ&agglutinin,&specific&for&
N-linked&polysaccharides,&is&used&to&identify&the&endomembrane&that&comprise&
the&Golgi&complex&
•
The&Golgi&complex&is&broken&down&into&the&cis-cisternae,&medial-cisternae&and&
trans-cisternae&
•
Proteins&in&the&Golgi&stay&in&the&cisternae,&and&the&cisternae&move&forward&
through&the&Golgi&complex&
•
Antibodies&attached&to&the&proteins&native&to&the&medial-cisternae&show&that&
these&proteins&are&mis-localized&and&have&to&be&transported&in&the&retrograde&
direction&via&vesicles&&
•
This&is&known&as&the&cisternal&maturation&model/cisternal&progression&model&•
New&cisternae&are&formed&by&coalescing&vesicles&from&the&ER&•
The&trans-Golgi&network&will&dissipate&into&secretory&vesicles&•
Steps&in&vesicular&trafficking&
There&are&3&types&of&coated&vesicles:&the&clathrin-coated&vesicles,&the&COP&I&
coated&vesicles&and&the&COP&II&coated&vesicles&
•
Clathrin-coated&vesicles&are&required&for&transport&away&from&the&trans-Golgi&
network&to&the&endosomes&and&to&the&cell&membrane&as&well&as&in&the&process&
of&endocytosis&&
•
COP&I&vesicles&are&used&specifically&for&retrograde&transport&from&the&Golgi&
back&to&the&ER&
•
COP&II&vesicles&are&required&for&transport&from&the&rough&ER&to&the&cis-Golgi&
network&
•
All&of&these&coat&proteins&are&small>P&binding&proteins&with>Pase&activity&•
Vesicles'form'by'a'process'called'budding;'buds'arise'from'the'membrane'of'
the'donor'compartment'
A>Pase&protein,&Sar1,&with&GDP&is&inactive&in&the&cytosol&
-
Sec&12,&a&transmembrane&protein&found&on&the&donor&compartment&of&
the&ER&is&a&guanine&exchange&factor&
-
Sar1&binds&Sec12&and>P&is&exchanged&for&GDP,>P&binding&induces&a&
conformational&change&in&Sar1&and&it&becomes&anchored&to&the&
membrane
-
COP&II&coat&proteins&(Sec23,&Sec24,&Sec13&and&Sec&31)&associate&with&
Sar1-GTP,&giving&the&complex&an&inherent&curvature&
-
Budding&in&the&Golgi&membrane&to&form&COP&I&and&clatharin-coated&
vesicles&occurs&the&same&way,&but&ARF&G-protein&is&used&instead&of&Sar1
-
1.
Cargo'proteins'are'loaded'into'buds'via'cargo'signal'sequences'and'receptors'
Cargo&receptors&accumulate&in&the&bud&and&pick&up&soluble&proteins,&
transmembrane&proteins&may&also&be&cargo&
-
Accumulation&of&the&cargo&proteins&is&possible&by&the&interaction&of&the&
cytosolic&domains&of&the&target&receptors&of&&transmembrane&cargo&with&
the&corresponding&specific&coat&proteins&
-
GTP&hydrolysis&converts&Sar1-GTP&into&Sar1-GDP,&releasing&it&from&the&
membrane&and&the&coat&proteins,&allowing&it&to&be&recognized&by&motor&
proteins&are&carried&alongµtubules&
-
Inhibiting>Pase&activity&results&in&the&prevention&of&docking&or&cargo&
unloading&
-
2.
Vesicles&formation&and&release
The&clathrin&coat&forms&a&polyhedral&lattice&or&a&tri-scallion&using&3&
clathrin&heavy&chains,&3&clathrin&light&chains&and&adaptor&proteins&
-
The&G&protein,&dynamin,&is&required&for&the&release&of&the&clatharin-
coated&vesicle&from&the&budding&membrane&
-
In&it's&active&form&it&is&bound&to>P&and&can&associate&with&the&neck&of&
the&budding&vesicle&
-
Dynamin&induces&vesicle& release&by&a&combination&of&the&pinchase&and&
poppase&models&
-
Using&a&fruit&fly&experiment,&if&dynamin&is&inhibited&then&there&can&be&no&
transport,&as&seen&with&the¶lyzed&flies&whose&neurotransmitters&
couldn't&be&transferred&due&to&a&restrictive& temperature&placed&on&a&
mutated&shibire&(which&codes&for&dynamin)&
-
3.
Vesicle&docking&and&fusion&to&membrane&of&the&recipient&compartment&
A&G&protein,&Rab-GDP,&is&free&is&the&cytosol&until&it&becomes&Rab-GTP&and&
is&bound&to&vesicles&by&a&hydrohpibic&anchor&
-
Rab-GTP&is&able&to&interact&and&bind&with&affinity&to&the&Rab&effector&to&
facilitate&docking&
-
This&isn't&enough&however,&the&vesicles&will&need&to&be&fused&together&
via&membrane-anchored&protein&helices& called&SNARES&
-
The&&vesicle&SNAREs&anchored&on&the&vesicle&membrane&is&VAMP&and&the&
target&SNARE&anchored&to&the&target&membrane&are&SNAP25&and&
syntaxin&
-
The&interaction&between&theses&SNAREs&mediates&fusion,&four&helices& 2&
from&SNAP25,&1&from&syntaxin&and&1&from&VAMP&spiral&together&to&form&
a&four&helix&bundle&
-
In&the&intermediate&state&the&SNAREs&are&pulled&apart&as&the&cytosolic&
domains&spiral&together,&a&hole&is&created&in&two&membranes&
simultaneously&so&that&the&contents&of&the&vesicles&don't&spill&out
-
After&fusion&and&the&release&of&the&cargo&contents,&the&SNARE&complex&
must&disassemble&so&that&the&proteins&can&be&reused&
-
NSF&and&the&alpha-snap&protein&associate&with&the&end&of&the&SNARE&
complex&and&unwind&the&4&helices&
-
The&SNARES&can&diffuse&in&the&membrane&for&recycling&and&reuse&once&
disassociated&&
-
4.
Module'5:'Lecture'2
Monday,&January&29,&2018
4:53&PM
Golgi&complex&
Proteins&travel&as&cargo&via&vesicles&from&the&ER&to&the&Golgi&complex&•
The&Golgi&complex&is&comprised&of&a&series&of&elongated&flat&sacs&called&
cisternae
•
Vesicles&transport&proteins&to&the&portion&of&the&cisternae&known&as&the&cis-
cisternae
•
Vesicles&leave&the&Golgi&complex&from&the&trans-cisternae,&there&are&two&
secretory&pathways:&
Constitutive&secretory&pathway
Used&by&proteins&that&are&released&immediately&after&protein&
synthesis&and&transport&
-
The&vesicles&can&fuse&with&the&cell& membrane&releasing&the&
protein&outside&the&cell&in&a&process&known&as&exocytosis&
-
I.
The®ulated&secretory&pathway&
Used&by&proteins&that&are&kept&in&the&cell&until&a&signal&triggers&
release&
-
The&secretory&vesicles&held&in&the&cell&are&called&secretory&granules&
-
II.
•
Other&proteins&carried&away&from&the&Golgi&trans-cisternae&in&vesicles&form&the&
lysosome&by&fusing&with&vesicles&at&the&cell& membrane&called&endosomes,&they&
capture&and&transport¯omolecules&from&outside&the&cell&
•
Instead&of&antibodies,&fluorescently-labeled&wheat&germ&agglutinin,&specific&for&
N-linked&polysaccharides,&is&used&to&identify&the&endomembrane&that&comprise&
the&Golgi&complex&
•
The&Golgi&complex&is&broken&down&into&the&cis-cisternae,&medial-cisternae&and&
trans-cisternae&
•
Proteins&in&the&Golgi&stay&in&the&cisternae,&and&the&cisternae&move&forward&
through&the&Golgi&complex&
•
Antibodies&attached&to&the&proteins&native&to&the&medial-cisternae&show&that&
these&proteins&are&mis-localized&and&have&to&be&transported&in&the&retrograde&
direction&via&vesicles&&
•
This&is&known&as&the&cisternal&maturation&model/cisternal&progression&model&
•
New&cisternae&are&formed&by&coalescing&vesicles&from&the&ER&
•
The&trans-Golgi&network&will&dissipate&into&secretory&vesicles&
•
Steps&in&vesicular&trafficking&
There&are&3&types&of&coated&vesicles:&the&clathrin-coated&vesicles,&the&COP&I&
coated&vesicles&and&the&COP&II&coated&vesicles&
•
Clathrin-coated&vesicles&are&required&for&transport&away&from&the&trans-Golgi&
network&to&the&endosomes&and&to&the&cell&membrane&as&well&as&in&the&process&
of&endocytosis&&
•
COP&I&vesicles&are&used&specifically&for&retrograde&transport&from&the&Golgi&
back&to&the&ER&
•
COP&II&vesicles&are&required&for&transport&from&the&rough&ER&to&the&cis-Golgi&
network&
•
All&of&these&coat&proteins&are&small>P&binding&proteins&with>Pase&activity&•
Vesicles'form'by'a'process'called'budding;'buds'arise'from'the'membrane'of'
the'donor'compartment'
A>Pase&protein,&Sar1,&with&GDP&is&inactive&in&the&cytosol&
-
Sec&12,&a&transmembrane&protein&found&on&the&donor&compartment&of&
the&ER&is&a&guanine&exchange&factor&
-
Sar1&binds&Sec12&and>P&is&exchanged&for&GDP,>P&binding&induces&a&
conformational&change&in&Sar1&and&it&becomes&anchored&to&the&
membrane
-
COP&II&coat&proteins&(Sec23,&Sec24,&Sec13&and&Sec&31)&associate&with&
Sar1-GTP,&giving&the&complex&an&inherent&curvature&
-
Budding&in&the&Golgi&membrane&to&form&COP&I&and&clatharin-coated&
vesicles&occurs&the&same&way,&but&ARF&G-protein&is&used&instead&of&Sar1
-
1.
Cargo'proteins'are'loaded'into'buds'via'cargo'signal'sequences'and'receptors'
Cargo&receptors&accumulate&in&the&bud&and&pick&up&soluble&proteins,&
transmembrane&proteins&may&also&be&cargo&
-
Accumulation&of&the&cargo&proteins&is&possible&by&the&interaction&of&the&
cytosolic&domains&of&the&target&receptors&of&&transmembrane&cargo&with&
the&corresponding&specific&coat&proteins&
-
GTP&hydrolysis&converts&Sar1-GTP&into&Sar1-GDP,&releasing&it&from&the&
membrane&and&the&coat&proteins,&allowing&it&to&be&recognized&by&motor&
proteins&are&carried&alongµtubules&
-
Inhibiting>Pase&activity&results&in&the&prevention&of&docking&or&cargo&
unloading&
-
2.
Vesicles&formation&and&release
The&clathrin&coat&forms&a&polyhedral&lattice&or&a&tri-scallion&using&3&
clathrin&heavy&chains,&3&clathrin&light&chains&and&adaptor&proteins&
-
The&G&protein,&dynamin,&is&required&for&the&release&of&the&clatharin-
coated&vesicle&from&the&budding&membrane&
-
In&it's&active&form&it&is&bound&to>P&and&can&associate&with&the&neck&of&
the&budding&vesicle&
-
Dynamin&induces&vesicle& release&by&a&combination&of&the&pinchase&and&
poppase&models&
-
Using&a&fruit&fly&experiment,&if&dynamin&is&inhibited&then&there&can&be&no&
transport,&as&seen&with&the¶lyzed&flies&whose&neurotransmitters&
couldn't&be&transferred&due&to&a&restrictive& temperature&placed&on&a&
mutated&shibire&(which&codes&for&dynamin)&
-
3.
Vesicle&docking&and&fusion&to&membrane&of&the&recipient&compartment&
A&G&protein,&Rab-GDP,&is&free&is&the&cytosol&until&it&becomes&Rab-GTP&and&
is&bound&to&vesicles&by&a&hydrohpibic&anchor&
-
Rab-GTP&is&able&to&interact&and&bind&with&affinity&to&the&Rab&effector&to&
facilitate&docking&
-
This&isn't&enough&however,&the&vesicles&will&need&to&be&fused&together&
via&membrane-anchored&protein&helices& called&SNARES&
-
The&&vesicle&SNAREs&anchored&on&the&vesicle&membrane&is&VAMP&and&the&
target&SNARE&anchored&to&the&target&membrane&are&SNAP25&and&
syntaxin&
-
The&interaction&between&theses&SNAREs&mediates&fusion,&four&helices& 2&
from&SNAP25,&1&from&syntaxin&and&1&from&VAMP&spiral&together&to&form&
a&four&helix&bundle&
-
In&the&intermediate&state&the&SNAREs&are&pulled&apart&as&the&cytosolic&
domains&spiral&together,&a&hole&is&created&in&two&membranes&
simultaneously&so&that&the&contents&of&the&vesicles&don't&spill&out
-
After&fusion&and&the&release&of&the&cargo&contents,&the&SNARE&complex&
must&disassemble&so&that&the&proteins&can&be&reused&
-
NSF&and&the&alpha-snap&protein&associate&with&the&end&of&the&SNARE&
complex&and&unwind&the&4&helices&
-
The&SNARES&can&diffuse&in&the&membrane&for&recycling&and&reuse&once&
disassociated&&
-
4.
Module'5:'Lecture'2
Monday,&January&29,&2018 4:53&PM
Document Summary
Proteins travel as cargo via vesicles from the er to the golgi complex. The golgi complex is comprised of a series of elongated flat sacs called cisternae. Vesicles transport proteins to the portion of the cisternae known as the cis- cisternae. Vesicles leave the golgi complex from the trans-cisternae, there are two secretory pathways: Used by proteins that are released immediately after protein synthesis and transport. The vesicles can fuse with the cell membrane releasing the protein outside the cell in a process known as exocytosis. Used by proteins that are kept in the cell until a signal triggers release. The secretory vesicles held in the cell are called secretory granules. Other proteins carried away from the golgi trans-cisternae in vesicles form the lysosome by fusing with vesicles at the cell membrane called endosomes, they capture and transport macromolecules from outside the cell. Instead of antibodies, fluorescently-labeled wheat germ agglutinin, specific for.
