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Lecture 6 Intracellular Compartments and Protein Sorting. Membrane-enclosed compartments. Proteins: enzymes, transporter and surface markers. 10,000-20,000 proteins delivered to different compartments. Major intracellular compartments.

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Presentation Transcript
slide1
Lecture 6
  • Intracellular Compartments
  • and Protein Sorting
slide2

Membrane-enclosed

compartments

Proteins:

enzymes,

transporter and

surface markers

10,000-20,000

proteins delivered

to different compartments

slide3

Major intracellular compartments

Vital chemical reactions take place in or on membrane surface

Compartments increase surface and isolate reactions

slide4

Microtubules help

the localization

of the ER and the

Golgi apparatus

Bacteria have no

Internal membranes

Eukaryotic cells are

1000-10,000 times

greater--need internal

membranes

slide8

Transport is highly regulated

Gated transport

Transmembrane transport

Vesicular transport

slide13

50 nucleoporins

Octagonal

Variable numbers of

pores (3000-4000)

depending on TXN

100 histone molecules

per minute per pore

6 large and small ribosomal

subunits per minute per pore

SEM

“Basket”

slide15

Ribosome 30 nm

DNA, RNA polymerases

100-200 Kd subunits

Results from injection:

<5000 Daltons: fast diffusion

17 Kd: 2 minutes

>60 Kd: cannot enter

Channel is 9 nm in diameter

15 nm long

slide16

An experiment using

recombinant DNA technique

One or two short sequences

Rich in positively charged aa

Lys, Arg

Immunofluorescence

micrographs showing

T-antigen localization

slide17

Gold particles coated

with nuclear localization

signals

Not through

lipid bilayer

Folded confomration

Pore dilates to 26 nm

Nuclear import

Receptors!!!

Bind to nucleoporins

FG-repeats

slide18

Nuclear export signals

Nuclear export receptors

Nuclear transport receptors (karyopherins)

A single pore complex conducts traffic in both directions

slide19

The Ran GTPase drives directional transport

Ran is required for both import and export

Asymmetical

Localizatin of GAP

And GEF!

GTPases are

molecular switches

GTPase-activating

Protein (GAP)

Guanine exchange

Factor (GEF)

slide20

Ran-GTP causes cargo

binding of export receptor

Ran-GTP causes cargo

release of import receptor

Free export receptors

return to the nucleus

GTP-bound import receptors return to the cytosol

slide22

The nuclear lamina

Meshwork of interconnected protein subunits, nuclear lamins

Intermediate filament proteins, interact with nuclear pore complexes

and integral membrane proteins, chromatin

slide23

NLS is not cleaved off

after transport--repeated

import

slide26

Mitochondrial proteins

are first fully synthesized:

different from proteins

transported into ER

Signal sequence:

Amphipathic a helix

Signal peptidase

slide28

Proteins transiently spanning the inner and outer membranes

during their translocation into the matrix

Precursor proteins remain unfolded

before transport

slide30

ATP hydrolyses at two sites plus

a H+ gradient across inner membrane

Release from cytosolic hsp 70

Further translocation through TIM requires H+ gradient

Release from mito hsp70

Signal peptide is positively charged

slide33

Two signal sequences

are required for proteins

directed to the thylakoid

membrane in chloroplasts

GTP and ATP

Signal sequences for

mito and chloroplasts

are different

Four routes

into the thylakoid

space

slide34

Peroxisomes have one single membrane

No DNA or ribosomes

Catalase and urate oxidase

Urate oxidase

Oxidative reactions not taken over by mito

RH2+O2->R+H2O2

Urate oxidate

(R=uric acid)

H2O2+R’H2->R’+2H2O

Catalase

b-oxidation

biosynthesis of plasmalogens

photorespiration

glyoxylate cycle

slide35

A model of how new peroxisomes are produced

From preexisting

peroxisomes

Transport mechanisms

unknown: no unfolding necessary

growth

23 peroxins

Similar to nuclear transport

fission

slide36

Summary

Cells are highly compartmentalized; proteins are sorted

to different compartments;

Nuclear transport, nuclear pore, nucleoporins, NLS;

Ran GTPase control direction;

Nuclear lamina, nuclear lamins;

Mitochondria transport, signal sequence, TOM, TIM,

energy;

Chloroplast transport, thylakoid;

Peroxisomes, structure, function, transport, biogenesis.