Advanced Cell Biology

2014 1nd Semester

Department of Animal Science

Chungbuk National University

5th Lecture

1st week : Introduction

3rd week :Research Strategies For Cell Biology

5nd week : Nucleus, Transcription and Splicing

7nd week : Membrane and Channel

9nd week : Membrane Trafficking

11nd week : Cell Signaling

13nd week : Cytoskeleton

15nd week : Cell Cycle

• Eukaryotic cells have an elaborate system of internal membrane-bounded structures called organelles.

• Each organelle:– has a unique composition of (glyco)proteins and

(glyco)lipids– carries out a particular set of functions

Why cell need Membrane Trafficking?

• An organelle comprises one or more membrane-bounded compartments.

• Organelles may act autonomously or in cooperation to accomplish a given function.

• In the endocytic and exocytic pathways, cargo proteins are transferred between compartments by transport vesicles.

Needs of Transport System..

• The vesicles form by budding from an organelle’s surface.

• They subsequently fuse with the target membrane of the acceptor compartment.

Vesicles

Exocytic pathway• All eukaryotes have the same complement of

core exocytic compartments: – the endoplasmic reticulum– the compartments of the Golgi apparatus– post-Golgi transport vesicles

Endocytic pathway• Extracellular material can be taken into cells by several

different mechanisms.

• The low pH and degradative enzymes in endosomes and lysosomes are important in processing some endocytosed material.

Endocytic and biosynthetic-secretory pathways

Transport vesicles

(Ten or more chemically distinct, membrane-enclosed compartments)

The biosynthetic-secretory and endocytic pathways

March 30, 2006 Pabio552, Lecture 2 13

A 2. ER translocation is co-translational

March 30, 2006 Pabio552, Lecture 2 14

March 30, 2006 Pabio552, Lecture 2 15

Please review the signal hypothesis and mechanisms of co-translational translocation in the Alberts’ textbook!

Vesicles

• Transport vesicles move proteins and other macromolecules from one membrane-bounded compartment to the next along the exocytic and endocytic pathways.

• Coats formed from cytoplasmic protein complexes help to:– generate transport vesicles – select proteins that need to be transported

• Proteins destined for transport to one compartment are sorted away from:– resident proteins– proteins that are destined for other compartments

• Transport vesicles use tethers and SNAREs to dock and fuse specifically with the next compartment on the pathway.

• Retrograde (backward) movement of transport vesicles carrying recycled or salvaged proteins compensates for anterograde (forward) movement of vesicles.

Various types of coated vesicles

Golgi apparatusPlasma

ER and Golgi Cisternae

COPII-coated vesicles : ER to Golgi

• ER membrane proteins activate Sar1p GTPase.

• Activate Sar1p bring togerther a transmembrane cargo receptor

• COPII coats deforms the membrane into a bud

• After budding, coat component promotes hydrolysis of GTP bound to Sar1p

COPI-coated vesicles : Golgi to the ER

• COPI coat assembly is triggered by a membrane-bound GTPase called ARF.

• ARF recruits coatomer complexes, and disassembly follows GTP hydrolysis.

• COPI coats bind directly or indirectly to cargo proteins that are returned to the endoplasmic reticulum from the Golgi apparatus.

Clathrin-coated vesicles : Golgi to endosome or

endocytosis

• COPI coat assembly is triggered by a membrane-bound GTPase called ARF.

• ARF recruits coatomer complexes, and disassembly follows GTP hydrolysis.

• COPI coats bind directly or indirectly to cargo proteins that are returned to the endoplasmic reticulum from the Golgi apparatus.

Assembly of a clathrin coat

triskelionCoated pitsand vesicleson the cytosolic surface of membranes

Freeze-etch

36 triskelions12 pentagons6 hexagons

Inner layer binds adaptins

Adaptin binds to cargo receptor and clathrin triskelion

Dynamin pinches of the vesicles

GTPase

Shibire mutanthas coated pitsbut no budding offof synaptic vesicles

ARF proteins: COPI&clathrinSar1 protein: COPII

GTP causes Sar1 toBind to membrane

Assembly and disassembly of coat by GTPases

Coat-recruitment GTPases GTPase works like a timerAnd cause disassembly shortlyAfter the budding is completed

Vesicle delivery : Rab GTPases as molecular ‘Zip’ Code

- Transport vesicle anchor one of 60 varieties of Rab

- Vesicle-associated GEF activate Rab

- Rab-GTP recruits tethering and fusion proteins

- After Membrane fusion, GAP activates and hydrolysis Rab-GTP

- Binds to GDI and return to donor membrane

SNARE proteins guide vesicular transport20 SNAREs, v-SNAREs, t-SNAREs

SNAREs specify compartment identity and control specificity

4 a helicesin trans-SNAREcomplexes

Rab proteins ensure the specificity of vesicle docking

>30 Rabs

On cytosolic surface

C-terminal regions are variable:Bind to other proteins, including GEFs

SNAREs may mediate membrane fusion

SNARE complex

After docking

The entry of enveloped viruses into cells

HIV

Similar to SNAREs

Proteins leave the ER in COPII-coated transport vesicles

ER exit sites(no ribosomes)

Selectiveprocess

Only properly folded and assembled proteins can leave the ER

Chaperones cover up exit signals

Homotypic membrane fusion

to form vesicular tubular clusters

The Golgi apparatus synthesizes sphingolipids, establishing a gradient of sphingolipids and cholesterol and bilayer thickness from low in the ER to high in the plasma membrane.

Transport through the Golgi apparatus: Membrane lipids, integral membrane proteins, and soluble proteins in the lumen move from the cis-Golgi through the stacks to the trans-Golgi.

An enzyme transfers the branched “core oligosaccharide” rich in mannose from dolichol to the side chain of asparagine (single letter abbreviation N) as the protein enters the lumen of the ER. An amide bond couples the first sugar to the side chain

Golgi apparatus glycosidases remove some sugars and glycosyltransferases add sugars to remodel oligosaccharide side chains.

ER retrieval signals: KKXX in ER membrane proteins,

KDEL sequence in soluble ER resident proteins

Membrane proteins in Golgi and ER have shorter TM domains (15 aa)Cholesterol

pH controls affinity of KDEL receptors

Targeting Protein and Membrane from TGN

The trans-Golgi network or TGN is a cluster of membrane-bounded tubules and vesicles adjacent to the trans-most stack of the Golgi apparatus.

Central sorting and distribution point for membranes and cargo coming through the secretory pathway and destined for lysosomes, endosomes, and plasma membrane.

Roles of Trans-Golgi Networks

Lysosome and Endosome

Endosomes : sorting compartments between the plasma membrane and lysosomes

Lysosomes contain a variety of hydrolytic enzymes that degrade proteins, lipids, polysaccharides, and nucleic acids taken into the cell by endocytosis (see next section) as well as many cellular molecules that turn over normally.

Endocytosis

- Cells utilize many different mechanism for endocytosis

- In phagocytosis and clathrin-mediated endocytosis, cell surface receptor selectively bind macromolegules to be internalized

Phagocytosis

Ingestion of large particles such as bacteria, foreign bodies, and remnants of dead cells

Four step in Phagocytosis

1. Attachments2. Engulfment

- Formation of phagocuytic cup- Growth of actin filaments

1. Fusion With Lysosome2. Degradation

Phosphatidylinositol (PO) is important regulator of Endocytosis

Macropinocytosis

- Many Cell ingest extercellular fluid in large endocytic structure called macropinosomes

Caveolae mediated Endocytosis

Caveole : enriched in cholestrolStabillized by the protein called caveolin

Histochemical stains: biochemicalCompartmentalization of the Golgi

Functional compartmentalization

Transport through the Golgi may occur by vesiculartransport or cisternal maturation (not mutually exclusive)

Collagen rodsScales in algae

Summary

1. Vesicular transport, biosynthetic-secretory andendocytic pathways;

2. Coated vesicles;3. Coat assembly and disassembly, budding, dynamin,

coat-recruitment GTPases;4. Targeting and fusion by Rab GTPases, SNAREs;5. ER to Golgi: COPII, folding, fusion (cluster), retrograde;6. Golgi apparatus structure and polarity;7. Continuation of glycosylation;8. Compartmentalization of Golgi cisternae;9. By now we have introduced gated transport, transmembrane

transport and vesicular transport.