大二下课件-第二部分intracellular

The

cell

cytoplasm

contains

many

different

specializedcompartmentsWhat

keeps

intracellular

trafficrunning

smoothly?Membranes

inside

cells

transport

key

nutrients

around,

andthrough,

various

cell

compartments

without

sticking

to

eachother

or

losing

their

way.Insights

into

how

membranes

stay

on

track

could

help

conquerdiseases,

such

as

cystic

fibrosis.囊泡

(traffic

jam)可导致许多重大疾病的发生，如白化病、神经退行性疾病、、肿瘤、

与免疫缺陷等。Three

distinct

mechanisms

utilizedNuclear

Pores

-Allow

the

transport

across

the

nuclear

membraneMembrane

transport

-

Allow

the

transport

across

theanelle

membraneVesicle

transport

-

Allow

the

transport

of

proteins

through

the

cell细胞内大分子的三种形式概括和比较an

important

difference

between

how

proteins

are

directed

to

theER

and

how

they

are

directed

to

othercytoplasmic

anelles:whereas

proteins

are

translocated

into

other anelles

onlyafter

their

synthesisis

complete,

they

are

translocated

into

theER

during

their

synthesis,

and

hencethe

ribosomes

on

whichthey

are

made

are

tethered

to

the

ER

membrane(1)

、The

protein

traffic

betweenthe

cytosol

and

nucleus

occursbetween

topologically

equivalent

spaces,

which

are

incontinuity

through

the

nuclearpore

complexes.This

processiscalled

gated

transport

because

the

nuclearpore

complexesfunction

as

selective

gates

that

can

actively

transport

specificmacromolecules

and

macromolecular

assemblies,althoughthey

also

allow

free

diffusion

of

smallermolecules.细胞内大分子

的三种形式概括和比较（续）、In

transmembrane

transport

membrane-bound

protein

translocators

directly

transport

specific

proteinsacross

a

membranefrom

the

cytosol

into

a

space

that

istopologically

distinct.The

transported

protein

moleculeusually

must

unfold

in

order

to

snake

through

themembrane.The

initial

transport

of

selected

proteins

fromthe

cytosol

into

the

ER

lumen

or

into

mitochondria,forexample,occurs

in

this

way.、

In

vesicular

transport,

transport

vesicles

ferry

proteinsfrom

onecompartmentto

another.

The

vesicles eloadedwith

a

cargo

of

molecules

derived

from

the

lumenof

one

compartment

as

they

pinch

off

from

itsmembrane;they

discharge

their

cargo

into

a

second

compartmentbyfusingwith

itsmembrane.Contents:2.3.1

introduction2.3.2

vesicular

transport2.3.3

Transport

from

the

ER

through

the

Golgiapparatus2.3.4

Transport

from

Golgi

destined

tolysosomes2.3.5

Transport

from

the

trans

Golgi

network

tothe

cell

exterior:

exocytosis2.3.6

Endocytosis2.3.1

Introductionthatto

theGolgienter

thereticulum

areandProteinsendoplasmictransportedtowards

theplasma

membrane.Specific

signals

cause

proteins

tobe

returned

from

the

Golgi

to

theER,

to

be

retained

in

the

Golgi,

tobe

retained

in

the

plasmamembrane,

or

to

be

transported

toendosomesProteins

maybetween

the

plasmaand

lysosomes.be

transportedmembraneand

endosomes.2.3.1

IntroductionSorting

signal

is

a

motif

in

a

protein(either

a

short

sequence

of

aminoacids

or

a

covalent

modification)that

is

required

for

it

to

beincorporated

into

vesicles

that

carryit

toa

specific

destination.2.3.1

IntroductionCoated

vesicles

are

vesicles

whose

membrane

has

on

itssurface

a

layer

of

a

protein

such

as

Clathrin,

COP-I

or

COP-II.Endocytosis

is

process

by

which

proteins

at

the

surface

ofthe

cell

are

internalized,

beingtransported

into

the

cell

withinmembranous

vesicles.Exocytosis

is

the

process

of

secreting

proteins

from

a

cellinto

the

medium,

by

transport

in

membranous

vesicles

fromoplasmic

reticulum,

through

the

Golgi,

to

storagevesicles,

and

finally

(upon

a

regulatory

signal)

through

theplasma

membrane.Retrograde

transport

describesmovement

of

proteins

in

thereverse

direction

in

the

reticuloendothelial

system,

typicallyfrom

Golgi

to

endoplasmic

reticulum.Contents:2.3.1

introduction2.3.2

vesicular

transport2.3.3

Transport

from

the

ER

through

the

Golgiapparatus2.3.4

Transport

from

Golgi

destined

tolysosomes2.3.5

Transport

from

the

trans

Golgi

network

tothe

cell

exterior:

exocytosis2.3.6

Endocytosis2.3.2

vesicular

transport2.3.2.1

Coated

vesicles

transport

bothexported

and

imported

proteinsProteins

are

transported

in

coated

vesicles.

Constitutive

(bulk

flow)transport

from

ER

through

the

Golgi

takes

place

by

COP-coatedvesicles.

Clathrin-coated

vesicles

are

used

for

both

regulated

exocytosisand

endocytosis.Coated

vesicles

transport

both

exported

andimported

proteinsVesicle

formation

resultswhen

coat

proteins

bindto

a

membrane,

deform

it,and

ultima y

surround

amembrane

vesicle

that

ispinched

off.Coated Coat

Proteins*

OriginVesicleClathrinTGNClathrin,

AP1,ARF1Clathrin,

AP2ClathrinPlasma

membraneCOP

ICOP

I,

ARF

1Bidirectional

transport

between

theER

and

Golgi

complex,

and

betweenGolgi

complex

cisternaeERCOP

IICOP

II

[Sec

13

andSec

23],

Sar1CaveolinCaveolinPlasma

membrane的衣被类2.3.2.2

Different

types

of

coated

vesicles

exist

in

each

pathway:膜泡型*ARF1

designates ribosylation

factor1（

-核糖基化因子1）;AP1

andAP2

designate

different

adaptor

protein

complexes

(also

called

assemblyprotein

complexes)1.

Clathrin-coated

pits

and

vesicles：Coated

vesicles

have

a

polyhedral

lattice

on

thesurface,

created

by

triskelions

of

clathrin.电镜The

structure

of

a

clathrin

coat13_1clathrin.movFor

more

information,

plsrefer

to

Kirchhausen

T’sworkClathrin

adaptor

proteins

(adaptins)衔接蛋白（adaptin）：介于笼形蛋白与配体受体复合物之间，起连接作用。目前至少发现4种不同类型的衔接蛋白，可分别结合不同类型的受体，形成不同性质的转运小泡，如AP1参与参与 基体→溶酶体的基体→内体的 、AP2参与质膜→内体的 、AP3。For

detailed

information

of

function

of

the

individual

subunits,

pls

referto

the

literatures,

i.e.,

Nature

Reviews

Neurosci,

2000,

Vol

1,

161.Cargo

recruitment

intoMechanisms

of

cargo

recruitmentinto

clathrin

coatedpits.Possible

interactions

of

adaptorproteins

with

cargo,

clathrin

and

AP-2

are

shown

by

broken

lines.Arrows

show

enzymatic

reactions

ofprotein

ubiquitinylation

andphosphorylation.Question

mark

indicates

that

themechanism

of

the

recruitment

ofendophilin-CIN85

complex

intocoated

pits

is

unclear.P:

phosphorylated

tyrosine,

serine

orthreonine

residues;

Ub:monoubiquitin;

X:

variable

residueThe

assembly/disassembly

of

clathrincoatcoatedpitscoatedvesiclesuncoatnucleationThe

role

of

dynamin

in

pinch

off

clathrin-

coated

vesicle

from

themembraneShibire

is

a

temperature

sensitive

phenotype

in

Drosophila

that

results

in

paralysisat

the

non-permissive

temperature

that

is

reversed

on

lowering

to

the

permissivetemperature.

The

phenotypeis

the

result

of

the

inability

of

endocyticvesicles

to

beseparated

from

the

parent

membranes

and

thus

there

is

a

depletion

of

vesiclesespecially

in

synaptic

terminals.Shibire

movieUncoating

of

the

vesiclesHsp70------ATPaseAuxillin-----activateATPaseHsc70

chrone

effects

the

uncoating

reaction,

and

isguided

to

appropriaocations

on

clathrin

lattices

by

theJ-

-containing

co-ch rone

molecule

auxilin.Death

of

a

Clathrin-Coated

Vesicle:Auxilin

binding

produces

a

local

change

inheavy-chain

contacts,

creating

a

detectablegolbal

distortion

of

the

clathrin

coat.Deathmov.movEndocytosis-celldance-smallSar1GTP酶与Sec23/Sec24复合体结合在一起，形成紧紧包围着膜的一层衣被，Sec13/Sec31复合体形成覆盖在

的一层衣被，Sec12是Sar1的鸟苷酸交换因子。2.

COPII-coated

vesicle

formation:Newly

synthesized

proteins

destined

for

secretion

are

sortedinto

COPII-coated

vesicles

at

specialized

regions

of

the

ER.3.

COP

I-coated

vesicle

formation:负责回收、转运内质网逃逸蛋白（esc

d

proteins）返回内质网。内质网通过两种机制维持蛋白质的平衡：一是转运泡将应被保留的驻留蛋白排斥在外，例些驻留蛋白参与形成大的复合物，因而不能被包装在出芽形成的转运泡中，结果被保留下来；二是通过对逃逸蛋白的回收机制，使之返回它们正常驻留的部位。内质网的正常驻留蛋白在C端含有一段回收信号序列（retrievalsignals），如果它们被意外地逃逸进入转运泡从内质网运至基体cis面，则cis面的膜结合受体蛋白将识别并结合逃逸蛋白的回收信号，形成COPI衣被小泡将它们返回内质网。Relationship

among

COPI,

COPII

and

clathrin

cagesSchematic

diagramcompares

the

vertex

geometry

of

COPI,COPII

andclathrin

cages.

The

inner

beta-propeller

sthat

form

vertex

contacts

are

drawn

as

orange

cyclinders,

theouter

-propeller s

as

yellow

cylinders,

and

the

-solenoid s

as

linked

green

hexagons.(The

hexagonsare

purely

schematic;

their

size

and

numberhave

nomeaning).膜泡

--衣被的形成衣被召集GTP酶（coat-recruitmentGTPase）：衣被召集GTP酶通常为单体GTP酶，也叫G蛋白，起分子开关的作用，结合GDP的形式没有活性，位于细胞质中，结合GTP而活化，转位至膜上，能与衣被蛋白结合，促进核化和组装。衣被召集GTP酶包括Arf蛋白和Sar1蛋白，Arf参与

基体上笼形蛋白衣被与COPI衣被的形成，Sar1参与内质网上COPII衣被的形成，两者的作用方式大体相似。质膜上笼形蛋白衣被的形成也与GTP酶有关，但其成分尚不明确。G蛋白具有两类重要的调节蛋白，即：鸟苷酸交换因子（guanine-nucleotide

exchange

fact

EF）和GTP酶激活蛋白（GTPaseactivating

protein,GAP）。GEF的作用是使G蛋白

GDP，结合GTP而激活。GAP的作用是激活G蛋白的酶活性，使GTP水解，G蛋白失活。2.3.2.3

膜泡

-定向机制衣被小泡沿着细胞内的微管被

到靶细胞器。各类小泡之所以能够被准确地和靶膜融合，是因为运输小泡表面的标志蛋白能被靶膜上的受体识别小泡1.SNARE

guides

vesicular

transport-介导特异性停泊与融合SNARE

structure

and

dissociation

oARE

complex位于靶膜上的叫作位于

小泡上的叫作v-SNAREs和t-SNAREs都具有一个螺旋结构域，能相互缠绕形成反式

SNAREs复合体NSF（N-ethylmaleimide-sensitive

fusion

protein,NSF）催化SNAREs的分离它是一种类似分子伴娘的ATP酶，能够利用ATP作为能量通过

几个适配蛋白（adaptor

protein）将SNAREs复合体的螺旋缠绕分开。破伤风毒素和肉毒素等细菌

的神经性毒素实际上是一类特殊的蛋白酶，能够选择性地降解SNAREs，从而阻断神经传导2.

Rab

proteins

help

ensure

the

specificity

of

vesicledocking-使

小泡靠近靶膜>

60

known

Rab

proteinsMonomeric

GTPaseIntracellular

distribution

with

specificityFacilitate

and

regulate

the

rate

of

vesicledockingSubcellular

Locations

of

Some

Rab

ProteinsRoles

of

Rab

proteinsContents:2.3.1

introduction2.3.2

vesicular

transport2.3.3

Transport

from

the

ER

through

the

Golgiapparatus2.3.4

Transport

from

Golgi

destined

tolysosomes2.3.5

Transport

from

the

trans

Golgi

network

tothe

cell

exterior:

exocytosis2.3.6

Endocytosis2.3.3

Transport

from

the

ERthrough

the

Golgi

apparatusGolgi

apparatusCarbohydrate

synthesisA

sorting

and

dispatchingsite

for

the

product

of

theERCis

Golgi

network,

transGolgi

network,

anizedwith

cis

to

trans

polaritySmall

vesicles

areassociatedAbundant

in

secretory

cellsRetention

ofERple y

assembled

antibody

molecules

in

theMask

the

ER

retention

signal,

such

as

Bip----for

these

resident

proteins,theERis

like

an

open

prison:thereis

nothing

to

stop

them

leaving,

but

if

they

leave,

they

are

broughtbackAnchor

the

proteinin

the

ER,

such

as

calnexinTransport

from

the

ER

to

the

Golgi

apparatus

is

mediated

by

vesicular

tubularclustersRetrieval

pathway

(from

Golgi)

to

the

ERContents:2.3.1

introduction2.3.2

vesicular

transport2.3.3

Transport

from

the

ER

through

the

Golgiapparatus2.3.4

Transport

from

Golgi

destined

tolysosomes2.3.5

Transport

from

the

trans

Golgi

network

tothe

cell

exterior:

exocytosis2.3.6

Endocytosis2.3.4

Transport

from

Golgi

destinedto

lysosomesSecretory

proteinsandproteins

of

the

vacuolarapparatus

andplasmalemma

aresynthesized

and

modifiedin

oplasmicReticulumSecretory

proteinsareprocessed

further

in

theGolgi

complexandthenexportedThe

best-understood

pathway

of

protein

sorting

in

the

trans

Golgi

networkNewly

synthesized

proteins

may

be

directed

to

late

endosomes(and

then

to

lysosomes)

from

the

Golgi

stacks.The

commonsignal

inlysosomal ing

is

the

recognition

of

mannose-6-phosphate（甘露糖-6-磷酸）

by

a

specificreceptor(M6PR).Lysosomes

are

small

bodies,

enclosed

bymembranes,

that

contain

hydrolytic

enzymes

ineukaryotic

cells.Three

pathways

to

degradation

inlysosomesScavenger

pathway—lysosomal

storagedisease（溶酶体储积病：由于溶酶体酶的遗传性缺陷而不能降解相应底物,导致该底物在溶酶体内沉积,引起细胞组织及

功能

）黏脂贮积症Ⅱ型乙酰氨基葡萄糖磷酸转移酶Ac-phosphotransferasemissing,

causing

defectedlysosomaling

signalContents:2.3.1

introduction2.3.2

vesicular

transport2.3.3

Transport

from

the

ER

through

the

Golgiapparatus2.3.4

Transport

from

Golgi

destined

tolysosomes2.3.5

Transport

from

the

trans

Golgi

network

tothe

cell

exterior:

exocytosis2.3.6

Endocytosis2.3.5

Transport

from

the

trans

Golginetwork

to

the

cell

exterior:

exocytosis（胞吐）Types

of

secretion（

）途Constitutive

secretory

pathway

(defaultpathway，组成型

途径)-Ubiquitous-Proteins

and

lipids

are

continually

secretedRegulated

secretory

pathway（调节型径）-Proteins

are

concentrated

and

stored

until

anextracellular

signal

stimulates

their

secretion-Hormone,

neurotransmitters,

or

digestive

enzymesThree

best

understood

secretory

pathwaysD1.

Secretory

vesiclesense-core

vesicles（DCV，致密囊泡）/

secretory

granules（颗粒）Clumps

of

aggregated,

electron-densematerials

are

seen

in

the

TGNThe

signal

for

aggregation

is

not

knownHow

to

know

there

is

a

signal

foraggregation?1.

囊泡向质膜附近转运；2.

囊泡与质膜栓系；3.

囊泡锚定在质膜上；4.

调节信号触发

囊泡与质膜融合；5.

囊泡与质膜融合从而

囊泡内的物质，即。Secretory

vesicles

wait

near

the

plasma

membraneuntil

signaled

to

release

their

contents-Very

fast-Not

all

vesicles

are

secreted-Signal

stimulated.Hormone.CalciumSecretory-vesicle

membrane

components

are

recycledAfter

discharge

on

the

plasma

membrane,

themembrane

components

of

the

vesicle

areremoved

from

the

PM

surface

by

endocytosisThis

removal

returns

the

proteins

of

thesecretory

vesicles

to

the

TGN,

where

they

canbe

used

again.Polarized

cells

direct

proteins

from

TGN

to

the

appropriateplasma

membraneof

theTwo

ways

of

sorting

plasma

membrane

proteins

in

a

polarized

epithelialcells2.Synaptic

vesicles（突触囊泡，小清亮囊泡）

can

form

directly

from

endocyticvesiclesSynaptic

vesicle-About

50

nm

of

diameter-Storage

of

neurotransmitter,

such

as

acetylcholine,

glutamate,GABASVs

are

not

assembled

in

the

TGN

network

but

in

theaxon

terminalSV

proteins

are

carried

to

endosomes

and

theplasmalemma

of

nerve

terminals

in

precusor

membranes.At

the

terminals

these

vesicle

precursorswould

joinexisting

SVs

as

they

pass

through

endosomes

during

therecyclingprocess.Synaptic

vesicles

can

form

directly

from

endocytic

vesiclesContents:2.3.1

introduction2.3.2

vesicular

transport2.3.3

Transport

from

the

ER

through

the

Golgiapparatus2.3.4

Transport

from

Golgi

destined

tolysosomes2.3.5

Transport

from

the

trans

Golgi

network

tothe

cell

exterior:

exocytosis2.3.6

Endocytosis2.3.6

EndocytosisPhagocytosis

(cellular

eating，吞噬)-Take

up

large

particles,

such

asmicro anismand

cell

debris-

Phagosome

（>250nm

dia.)Pinocytosis

(cellular

drinking，胞饮)-

Take

up

fluid

and

solute-

Small

vesicles

(<150nm

dia.)PhagocytosisTake

up

nutrientProtection---macrophages

（巨噬细胞）

,neutrophils

（中性粒细胞）and

dendritic

cells（树突状细胞）Scavenger-phagocytose>1011

rbc(血红细胞)/day

by

macrophagePinocytosisClathrin-coated

pitsnonclathrin-coated

vesiclesPinocytosisMacrophages

pinocytose

25%

of

its

own

volume

each

day.

This

translates

into3%

of

its

membrane

surfaceare r

minute,

or

100%

per

0.5

hour.Principally

mediated

via

clarthrin-coated

pits.

Typically

occupy

about

2%

of

thetotal

surface

area.

It

has

been

estimated

that

more

than

2,500

clathrin-coated

pitsare

internalized

ea inute

in

a

normal

fibroblast.

This

is

a

constitutive

process.Coats

only

last

for

at

most

one

minute;

within

seconds

of

being

internalized

theylose

their

membrane

coats

and

fuse

withearly

endosomes.Extracellular

fluid

is

trapped

withincoated

vesicles

as

they

invaginate

to

formvesicles

-

hence

“cell

drinking”.

Also

known

as

fluid

phase

endocytosis.Deeply

invaginated

structures

formed

in

patches

of

membrane

known

as

lipid

rafts.Major

components:

Cholesterol,

glycosphingolipids,

GPI-anchored

proteins

anda

protein

known

as

caveolin.In

contrast

to

classes

of

protein

coated

vesicles,

caveolae

are

thought

to

invaginateand

collect

cargo

by

virtue

of

their

membrane

composition,NOT

PROTEINCOAT.Receptor-mediated

endocytosis>25

receptorsLDL（低密度脂蛋白）

receptorTransferin（转铁蛋白）

receptorEGF（表皮生长因子）

receptorLDL

receptor

recyclingLDL与LDL

受体在弱酸性的早期内涵体中分离，LDLR通过循环内涵体重新返回质膜，而

LDL则进入溶酶体，消化，转变为胆固醇分子，进而参与更多的生理过程。Transferin

receptor

recyclingCell

surface—Receptor-transferin-iron---Endosome----

Receptor-apotransferrin—recycling-----apotransferrin（脱离了Fe3+的转铁蛋白）released

from

the

receptorApotransferrin

and

receptor

donot

reach

lysosomeEarly

endosomeEGF

receptor

down

regulationEGF

receptor

is

degraded

in

the

lysosomeinstead

recycling

----down

regulationThe

sequestration

of

endocytosed

proteins

into

internal

membranes

of

MVBMultivesicular

bodies（

泡体）Fates

of

receptors

after

endocytosisTranscytosis(转胞吞)→Polarized

cellsTight

junctionApical--Rich

in

glycolipidBasolateralStorage

of

plasma

membrane

proteins

in

recycling

endosomesSummaryProteins

that

reside

within

the

reticul