• Lecture 6

• Intracellular Compartments

• and Protein Sorting

Membrane-enclosedcompartments

Proteins:enzymes,transporter andsurface markers

10,000-20,000proteins deliveredto different compartments

Major intracellular compartmentsVital chemical reactions take place in or on membrane surface

Compartments increase surface and isolate reactions

Microtubules helpthe localizationof the ER and theGolgi apparatus

Bacteria have noInternal membranes

Eukaryotic cells are 1000-10,000 timesgreater--need internalmembranes

Development ofplastids

topology

Topologically equivalent spaces are shown in red

Gated transport

Transmembrane transport

Vesicular transport

Transport is highly regulated

Signal sequence and signal patch

50 nucleoporinsOctagonal

Variable numbers ofpores (3000-4000)depending on TXN

100 histone moleculesper minute per pore

6 large and small ribosomalsubunits per minute per pore

SEM

“Basket”

Results from injection:

<5000 Daltons: fast diffusion17 Kd: 2 minutes>60 Kd: cannot enter

Channel is 9 nm in diameter15 nm long

Ribosome 30 nm

DNA, RNA polymerases100-200 Kd subunits

One or two short sequencesRich in positively charged aaLys, Arg

Immunofluorescencemicrographs showing T-antigen localization

An experiment usingrecombinant DNA technique

Gold particles coatedwith nuclear localizationsignals

Pore dilates to 26 nm

Not throughlipid bilayer

Folded confomration

Nuclear importReceptors!!!

Bind to nucleoporins

FG-repeats

Nuclear export signalsNuclear export receptors

Nuclear transport receptors (karyopherins)

A single pore complex conducts traffic in both directions

The Ran GTPase drives directional transport

Ran is required for both import and export

GTPases aremolecular switches

GTPase-activatingProtein (GAP)

Guanine exchangeFactor (GEF)

AsymmeticalLocalizatin of GAPAnd GEF!

Ran-GTP causes cargorelease of import receptor

Ran-GTP causes cargobinding of export receptor

Free export receptorsreturn to the nucleusGTP-bound import receptors return to the cytosol

Nuclear localization of TXN factors control gene expression

The nuclear lamina

Meshwork of interconnected protein subunits, nuclear lamins

Intermediate filament proteins, interact with nuclear pore complexesand integral membrane proteins, chromatin

NLS is not cleaved offafter transport--repeatedimport

The subcompartments of mitochondria and chloroplasts

Mitochondrial proteinsare first fully synthesized:different from proteinstransported into ER

Signal sequence:Amphipathic helix

Signal peptidase

Translocases of the outer and inner mito membranes

Proteins transiently spanning the inner and outer membranesduring their translocation into the matrix

Precursor proteins remain unfoldedbefore transport

Protein import by mitochondria

ATP hydrolyses at two sites plus a H+ gradient across inner membrane

Release from cytosolic hsp 70

Further translocation through TIM requires H+ gradient

Signal peptide is positively charged

Release from mito hsp70

Two models of how mito hsp70 could drive protein transport

Proteins destined for various mito space

Two signal sequencesare required for proteinsdirected to the thylakoidmembrane in chloroplasts

Four routesinto the thylakoidspace

Signal sequences formito and chloroplastsare different

GTP and ATP

Urate oxidase

Peroxisomes have one single membrane

No DNA or ribosomes

Catalase and urate oxidaseOxidative reactions not taken over by mito

RH2+O2->R+H2O2

H2O2+R’H2->R’+2H2OCatalase

Urate oxidate(R=uric acid)

-oxidation

biosynthesis of plasmalogens

photorespiration

glyoxylate cycle

A model of how new peroxisomes are produced

From preexistingperoxisomes

growth

fission

Transport mechanismsunknown: no unfolding necessary

23 peroxinsSimilar to nuclear transport

Summary

1. Cells are highly compartmentalized; proteins are sortedto different compartments;

2. Nuclear transport, nuclear pore, nucleoporins, NLS;3. Ran GTPase control direction;4. Nuclear lamina, nuclear lamins;5. Mitochondria transport, signal sequence, TOM, TIM,

energy;6. Chloroplast transport, thylakoid;7. Peroxisomes, structure, function, transport, biogenesis.