RNA localization

RNA localizationmRNA can be localized to subcellular compartmentsby actin or tubulin-dependent processesExamples:

Xenopus: Vg1 mRNA (TGF) to vegetal pole

Drosophila: nanos, oskar mRNA (posterior) and bicoid (anterior)

(requires mRNA binding protein staufen)

(requires staufen and miranda)

prospero (into ganglion of mother cells; neuroblast TF)

Yeast: Ash1 mRNA to daughter cell

lamellipodia staining perinuclear staining in myotubes

Bertrand et al., Mol Cell (98) 2:437-445

SUMMARY 2

I. mRNA decay- regulated and non-regulated turn-over- ordered pathway (deadenylation, decapping, exonucleolytic degradation)- NMD: recognition of premature stop codons

II. Cytoplasmic mRNA localization- ZIP code in 3’ UTR- both actin and tubulin-mediated - yeast mating type switch as a model: Ash1 mRNA localization (via 3’ UTR, She2/3, Myo4 and actin cables)

ER translocation & vesicular transport

from: Jamieson and Palade

3min 7min

37min 117min

N

N

N

O

N

NH2

H

N

N

N

N H

O

O

HN Ala 151

HN Ala 151

O

O

C Asp 125

Val 124

Lys 123

Asn 122

NH3+

O

H2N

C Asn 125

Val 124

Lys 123

Asn 122

NH3+

Ran WTRan WT

Ran D125NRan D125N

GTPGTP

XTPXTP

In vitro reconstitution of ER translocation:

- Sec61 complex: conserved translocation channel Sec61 subunits () Sec62/63 TRAM (translocating chain-assoc. membrane protein)

- phospholipids (proteoliposomes) and luminal chaperones (BIP)

- SRP/SRP receptor only required for co-translational translocation not for post-translational translocation (e.g pre-pro-alpha factor).

- energetics of translocation: protein conducting channel (cotranslational) molecular ratcheting (posttranslational)

Probing of translocation intermediates with fluorescent peptides

From: Liao and Johnson Cell (97)

The Sec61 complex forms a channel

Menetret et al. Mol Cell (2000) 6:1219

From: Beckmann et al. Cell (2001) Vol 107, 361-372

From: Beckmann et al. Cell (2001) Vol 107, 361-372

From: Van den Berg et al. Nature (2004) 427, 36-44

From: Van den Berg et al. Nature (2004) 427, 36-44

Topology of membrane-spanning proteins

Type I membrane proteins have a cleavable signal sequence

Type II membrane proteins have internal signal sequence

Type III membrane proteins have internal signal sequence

Type II+III membrane proteins have internal signal sequences

From: Beckmann et al. Cell (2001) Vol 107, 361-372

Translocation of proteins with multiple membrane spanning domains

From: Van den Berg et al. Nature (2004) 427, 36-44

Formation of a glycosylphosphatidylinositol (GPI)-anchor

ER function

- Proper folding of proteins (chaperones, lectins, petidyl-prolyl-isomerases)- Formation of disulfide bonds (PDI) GSH prevents oxidation in cytosol GS-SG + NADPH + H+ <=> 2 GSH + NADP+

- Proteolytic cleavages- Addition & processing of carbohydrates- Assembly into multimeric proteins

- Ca2+ storage- Lipid synthesis- Detoxification (liver!)

Folding of Influenza hemagglutinin (HA)

Ser/Thr

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Summary

ER translocation: SRP-dependent and -independent pathways; translocation occurs through Sec61 complex; topogenic sequences determine overall orientation.

ER function

Compartmental identity: maturation versus fixed compartments

Identification of components: combination of genetics, biochemistry...

Vesicular coats: COPI ~ retrograde: Golgi->ER COPII ~anterograde: ER->Golgi CCV post-Golgi, various adaptors