Er to golgi transport
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ER-to-Golgi Transport. Pathway Maturation De Novo Golgi Biogenesis Mitotic Assembly/Disassembly Post-ER Processing. Pathway Diagram. Sorting, Retention, Retrieval. ER. ERGIC. Golgi. The Golgi is the central processing and sorting station of the secretory pathway. ER. ERGIC. Golgi.

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ER-to-Golgi Transport

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Er to golgi transport

ER-to-Golgi Transport

Pathway

Maturation

De Novo Golgi Biogenesis

Mitotic Assembly/Disassembly

Post-ER Processing


Pathway diagram

Pathway Diagram


Sorting retention retrieval

Sorting, Retention, Retrieval


Er to golgi transport

ER

ERGIC

Golgi

The Golgi is the central processing and sorting station of the secretory pathway

ER

ERGIC

Golgi


Live imaging of vsvg gfp transport

Live Imaging of VSVG-GFP Transport


Copii mediates er export

COPII Mediates ER Export


Homotypic fusion copii vesicles may form ergic vtcs

Homotypic fusion COPII vesicles may form ERGIC/VTCs


Vtc morphology

VTC morphology


Ergic vtc moves along microtubules

ERGIC/VTC moves along microtubules

-

+


Er to golgi transport

The Golgi maintains its complex structure in the presence of continuous membrane traffic

trans

medial

cis


3d view of golgi stack

3D View of Golgi Stack


By what mode does golgi traffic occur

By what mode does Golgi traffic occur?


Vesicle maturation models

Vesicle & Maturation Models


Enzymes in copi

Enzymes in COPI?

Table II. Relative lateral distribution of Golgi proteins and VSV-G within the Golgi complex

Golgi stack (cisternae)Lateral rims (uncoated)Coated buds and vesicles

Man II68.9 ± 3.913.6 ± 2.717.5 ± 2.8

Giantin58.2 ± 2.618.3 ± 2.823.5 ± 2.7

KDELr35.8 ± 4.38.9 ± 2.055.3 ± 4.5

rBet125.6 ± 4.915.7 ± 4.058.7 ± 5.6

VSV-Gts045–GFP (20 min, 32°C)

anti-GFP93.1 ± 1.44.3 ± 1.32.6 ± 0.6

anti–VSV-G–lum

94.1 ± 0.83.4 ± 0.62.5 ± 0.5

------------------------------------------------------------------------

Numbers represent the percentages (mean ± SEM) of the total labeling over the distinct membrane

categories and were obtained by analyzing 50 Golgi complexes for each antibody.


Enzymes not in copi

Enzymes not in COPI?


Copi vesicles concentrate enzymes not cargo

COPI Vesicles Concentrate Enzymes not Cargo

------------------------------------------------------------------------

MarkersAmount of vesicles (% of starting membranes)

------------------------------------------------------------------------

GTPGTPSarf-1 Q71L

------------------------------------------------------------------------

Golgi enzymes

Mann II13.2 ± 1.36.1 ± 1.44.2 ± 1.1

NAGT I25.8 ± 2.73.1 ± 1.03.0 ± 0.8

GalT11.0 ± 2.01.7 ± 0.41.8 ± 0.4

Anterograde cargo

pIgR 4.6 ± 1.03.6 ± 1.43.3 ± 1.1

Phospholipids 2.7 ± 0.73.1 ± 0.72.5 ± 0.8

------------------------------------------------------------------------


How is the golgi formed

How is the Golgi Formed?


Trafficking may be sufficient for golgi self assembly

Trafficking may be sufficient for Golgi self-assembly

Golgi

ERGIC

cis

medial

trans

Trans Golgi Network

Homotypic fusion

MT

COP I

COP I

COP I

ER

COP II


Can the golgi self assemble

Can the Golgi self-assemble?

Idea: Use a reversible ER export block

-Brefeldin A to collapse Golgi

-H89 to block ER export and collapse ERGIC


Collapse of matrix non matrix markers into er

Collapse of matrix & non-matrix markers into ER


Gm130 co fractionates with er membranes

GM130 co-fractionates with ER membranes

Golgi

ER

untreated

BFA H89


Re emergence of the golgi from the er

min after H89 washout

0’

4’

Re-emergence of the Golgi from the ER

GM130

10’

30’


Reassembled golgi membranes are stacked

Reassembled Golgi membranes are stacked

BFA H89

30’ washout


Early compartmentalization of golgi membranes after er exit

Early compartmentalization of Golgi membranes after ER exit

5’

Giantin

GPP130


Initial stages in de novo golgi biogenesis

Initial Stages in “de novo” Golgi biogenesis


De novo biogenesis continued

De novo biogenesis continued


Transport paths consequences of their regulation

Transport paths & consequences of their regulation

Mitotic: vesicles=>block in docking

Osmotic:ER=>ER export block


M phase golgi vesiculation

M-phase Golgi Vesiculation


Tethered vesicle transport inhibited at m phase

Tethered vesicle transport: inhibited at M-phase?


Er to golgi transport

Anti-p115 injection induces dispersion of the Golgi in vivo


Er to golgi transport

Blocking p115, but not giantin or GM130, causes vesiculation


Er to golgi transport

What are the essential p115 interactions?

(i.e. candidates for regulation at mitosis)

acidic tail

head domain

coiled-coil domain

Rab1

Giantin GM130

PLCg1

SNAREs

phosphorylation site

GBF1


Beauty is in the rnai of the beholder

Beauty is in the RNAi of the Beholder


Er to golgi transport

100

80

60

40

mock

24 h

48 h

72 h

96 h

20

0

mock

96 h

p115 siRNA induces time-dependent depletion of p115

GPP130

p115

p115

mock

%intact

p115 levels

siRNA


Er to golgi transport

siRNA-induced p115 depletion results in Golgi fragmentation

p115

GPP130

mock

siRNA


Er to golgi transport

20

40

60

80

100%

f

+p115

Mismatched p115 rescues siRNA-induced Golgi fragmentation

inj. marker

p115

GalNac-T2

% injected cells with intact Golgi


Er to golgi transport

O

The giantin/GM130-binding domain is NOT required for Golgi biogenesis in vivo

WT

S941A

Dtail

Acidic Tail

20

40

60

80

100%

Targeted?

+

WT

+

S941A

+

Dtail

% expressing cells with intact Golgi


Er to golgi transport

O

WT

Dcc1

The SNARE domain of p115 is required for Golgi biogenesis in vivo

cc1

+

+

+

+

+

?

+

Targeted?


Er to golgi transport

Working model for mechanism of action of p115

recruitment of p115 by Rab1

binding to

t-SNARE

catalysis of SNARE pairing

p115

v-SNARE

Rab1

t-SNARE

=> dissect SNARE mechanism w/ further mutagenesis

=> test other p115 interactions-> distinct phenotypes?


Er to golgi transport

rbet1

syntaxin5

membrin

sec22

Sub-regions of the cc1 domain show homology to distinct SNAREs

rbet1

cc1

core complex domain

syntaxin5

membrin

p115 homology region

sec22


Part 1 post er processing

Part 1 Post-ER Processing

Modification of carbohydrate side-chains

Proteolytic activation of precursors


Er to golgi transport

Compartmentalization of processing reactions


Er to golgi transport

Branched carbohydrate


Er to golgi transport

Evidence for enzyme compartmentalization


Er to golgi transport

Processing Reactions

endoH sensitive

endoH resistant

cis

trans


Example of proteolytic activation

Example of proteolytic activation


Proton pumps reduce lumenal ph

Proton pumps reduce lumenal pH


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