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
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