Chapter 12

• Intracellular Compartments and Protein Sorting

張學偉 助理教授

The compartmentalization of cells

All eucaryotic cells have the same basic set of membrane-enclosed

organelles

The major intracellular compartments of an animal cells.

Cytoplasma = cytosol + cytoplasmic organelles

The topological relationships of membrane-enclosed organelles

can be interpreted in terms of their evolutionary origins

Protein can move between compartments in different ways

Sorting signal by signal sequences

Vesical transport

Signal sequences and signal patches direct proteins to the

correct cellular address

Sorting signal (signal sequences) recognize by sorting receptors

Cut by signal peptidases

Red +Green -Yellow HydrophobicBlue hydroxylated

N-terminal signalC-terminal signal

The transport of molecules between the nucleus and the

cytosol

Nuclear pore complexes perforate the nuclear envelope

Composed by more than 50 different proteins called nucleoporins.

9nm

26nm15nm

size

Nuclear localization signals (NLS) direct nuclear proteins to the

nucleus

Colloidal gold spheres coated with peptides containing NLS

Nuclear pore transport (large aqueous pore) is fundmental different from organelle transport (lipid bilayer).

Nuclear import receptors bind nuclear localization signals and

nucleoporins

FG-repeat (Phe-Gly) serve as binding sites for the import receptors.

Solublecytosolicprotein

Nuclear import do not always bind to nuclear proteins directly.

Nuclear export works like nuclear import, but in reverse

Nuclear export signals & nuclear export receptor & nuclear transport receptor (karypherins)

tRNA or 5S RNA: nuclei cytosolNLS-particle: cytosol nuclei

The Ran GTPase drives directional transport through

nuclear pore complexes

Ran = GTPaseGAP = GTPase-activing proteinGEF = Guanine exchange factor

Bidirectional model

Transport between the nucleus and cytosol can be regulated by

controlling access to the transport machinery

Always in & out, shuttling

Ventral side Dorsol protein

The control fly embryo development by nuclear transport

The nucleus envelope is disassembled during mitosis

Lamina (whole structure) & lamins (protein subunit)

The transport of proteins into mitochondria and chloroplasts

Newly mito & chloropl are produced by the growth of preexisting organelle.Their growth depends mainly on the cytosolic protein import

Translocation into the mitochondrial matrix depends on a

signal sequence and protein translocators

Red = +Yellow = nonpolar

On different side

Amphipathic helix

translocase

Require for import all nucleus-encoded mitochondria protein

Insert to inner memb.Transport to matrix

For protein synIn mito

Mitchondrial precursor proteins are imported as unfolded

polypeptide chains

Interacting protein: eg Charperone protein hsp70 family

All Interacting protein help to prevent aggregation before engaging with TOM complex in outer mito membrane.

Mitochondrial precursor proteins are imported into the matrix at contact sites that join the inner

and outer membranes

Protein import by mitochondria

ATP hydrolysis and a H+ gradient are used to drive protein import

into mitochondria

pulling

Freely permeable to ions and metabolitesbut not to most protein

Charperone protein also function as translocator

Repeated cycles of ATP hydrolysis by mitochondrial Hsp70

complete the import process.

Hsp 60 provide chamber for unfolded polypeptide chain facilitates folding (chapter 6)

Protein transport into the inner mitochondrial membrane and the intermembrane space required

two signal sequences

Two signal sequences are required to direct proteins to the

thylakoid membrane in chloroplasts

Resemble in mitochondria

peroxisomes

Peroxisomes use molecular oxygen and hydrogen peroxide to

perform oxidative reactions

Catalase: 2H2O2 2H2O + O2Urate oxidase: RH2 + O2 R + H2O2

Animal: -oxidation occur at both mitochondria & perixosome.

Plant & yeast: -oxidation occur only at perixosome.

Plasmalogen-the most abundant protein in myelin.- deficient result in neurological disease.

Animal Perxisome catalyze the first step for plasmalogen biosyn

Glyoxylate cycle

A short signal sequence directs the import of proteins into

peroxisomes

Peroxins:-at least 23 distinct proteins for driving ATP hydrolysis-deficent result in Zellweger syndrome.

Most peroxisomal membrane proteinsare made in the cytosol insert into preexisting peroxisomes.

The endoplasmic reticulum

Membrane-bound ribosomes define the rough ER

Many ribosomes bind to a single mRNA

ER capture 2 type of protein: transmembrane protein & water-sol protein

Cotranslatioal transport?Posttranslational transport?

p690

In mammalian cellsProtein import to ER Cotranslational process (chaperone are not required to keep protein unfolded)Protein import to mitochondria, chloroplasts, nuclei, peroxisomes Postranslational process (chaperone needed for unfolding)

Compared to page 697

Smooth ER abundant in some specialized cells

Lipid metabolism (cholestersol)Detoxification by cytochrome p450Sequester Ca+2 from cytosol (SR)

Autophagocytosis & phenobarital

Rough and smooth regions of ER can be separated by

centrifugation

Cell-free system

Signal sequences were first discovered in proteins imported

into the rough ER

A signal-recognition particle (SRP) directs ER signal sequences to a specific receptor in the rough ER

membrane

ER & SRP for import

The polypeptide chain passes through an aqueous pore in the

translocator

Translocation across the ER membrane does not always

require ongoing polypeptide chain elongation

p693

rare

yeast

ATPase

Binding protein(hsp70-like chaperone protein)

Protein that areare first released into cytosol (bind to hsp to prevent folding)

c/o sealing the pore

The ER sequence is removed from most soluble proteins after

translocation

Start-transfer signal

In single-pass transmembrane proteins, a single internal ER

signal sequence remains in the lipid bilayer as membrane-

spanning of a helix

Combinations of start-transfer and stop-transfer signals determine

the topology of multipass transmembrane proteins

hydrophobicity

Translocated polypeptide chains fold and assemble in the lumen of

the rough ER

Important ER resident proteins: PDI (protein disulfide isomerase; produce -s-s-)BiP chaperone protein (prevent aggregate & help to keep in ER)

Most (Soluble & membrane-bounded) proteins synthesized in the RER are glycosylated by the addition of a common N-linked

oligosaccharide

Very few protein in cytosol is glycosylated.

N-linked oligosaccharide - are by far the most common oligosaccharides found in glycoprotein. (RER)-are recognized by 2 ER charperon protein (calnexin & calreticulin)

O-linked oligosaccharide are found in Golgi.

Oligosaccharides are used as tags to mark the state of protein

folding

Improperly folded proteins are exported from the ER and

degraded in the cytosol

deglycosylation

Retrotranslocation(dislocation)

Misfolded proteins in the ER activate an unfolded protein

response

Some membrane proteins acquire a covalently attached

glycosylphosphatidylinositol (GPI) anchor

Segregate protein from other membrane protein

Most membrane lipid bilayers are assembled in the ER

Phospholipid exchange proteins help to transport phospholipids

from the ER to mitochondria and peroxisomes

1. Roadmap of protein traffic2. Signal sequences & organelle targeting3. Organelle epigenetic control

4. Nuclear pore complex & nuclear import/export & its receptor/signal5. The control of nuclear import during T-cell activation

6. Protein translocation process in mitochondrial membrane: TOM, TIM, OXA7. Relationship among import of mitochondrial precursor proteins, role of energy,its

hsp70.8. Translocation of a precursor protein into the thylakoid space of chloroplasts.

9. Peroxisomal enzymes & reactions, import mechanism distinct from mitochondria & chloroplast or unique character of peroxisome

10. SER, RER preparation, SRP, ribosome and RER protein transport11. Cotranslation & postranlation translocation in bacteria, archea, and eucaryotes12. Hydrophobicity of membrane protein and transmembrane domain13. Process and role of protein N-link glycosylation in RER14. Membrane lipid bilayer assembly in ER: using example of phosphatidylcholine

synthesis15. Phospholipid transport from ER to other organelles and comparison of ER and plasma membrane

Chapter 12 practice