BIOL2011: Protein targeting and export –

Lecture 1
Dr Mark Coldwell
Life Sciences Building 85, 3051 M.Coldwell@soton.ac.uk

 These lectures are designed to give you an introduction to:

 Signals in proteins and mRNAs that determine how proteins go to the correct point in the
cell
 The machinery needed to accomplish these localisations
 How proteins are exported from the cell

 In addition to learning the theory, I expect you to gain an understanding of some of the
laboratory techniques used to provide evidence for the models presented

 These lectures are only the start – I expect you to read further using textbooks: Lodish –
Molecular Cell Biology; Alberts – Molecular Biology of the Cell; Stryer – Biochemistry
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Overview

Lodish 7th Edition, Fig 13-1

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Protein targeting and export
 How do the 33,000+ different proteins in a eukaryotic cell find their correct cellular
location with over 30 possible target sites?
– cytoplasm
– nucleus
– plasma membrane
– cell exterior
– smooth and rough endoplasmic reticulum
– Golgi, lysosomes, peroxisomes
– mitochondrial and chloroplast compartments,
– vacuoles, glyoxisomes, periplasmic space...

Why are these pathways needed?

 Average protein is 10 nm across = 41 nm3 volume

 Average eukaryotic cell, 50 µm dia = 6.6x1013 nm3 volume
 Random diffusion v “Energetic” process?
 What if a protein is located in the incorrect compartment?
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Cellular logistics
1) Origin and destination?
2) What is the nature of the cargo?
3) What “barrier(s)” needs to be passed through?
4) What specific signal is needed?
5) What recognises this signal?
6) How is transport achieved?
 one step or a chain of events?
 one piece of cargo at a time, or multiples?

7) Post-transport events?
 Cargo: ensuring one-way traffic
 Returning transporters to original site
 Using fluorescent proteins to visualise
5

subcellular localisation
 GFP from Aequorea victoria (Nobel prize winning)
 Other colours can be obtained by mutating the b-barrel structure (YFP, BFP, CFP)
or using proteins from other organisms (DsRed)
 Can make fusions with your protein of interest (chimera)
 Disadvantages: chimeric protein may not fold correctly/act normally due to large
size of FP

http://en.wikipedia.org/wiki/Green_Fluorescent_Protein
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Using immunofluorescence (IF)
 Same kind of procedure as immunoblotting
 Disadvantages:
 Cells have to be fixed and permeabilised to allow antibody into them i.e. Dead!
 Antibodies may give false signals via non-specific binding

Specific antibodies Allow antibodies

against antigen to bind to antigen
Antibodies labelled (primary antibody)
with fluorescent dye
Allow antibodies
to bind to antigen
Specific antibodies
against antigen

Add labelled antibodies

that bind to primary
antibody (“secondary
(a) Immunofluorescence antibody”)

b) Indirect Immunofluorescence

Becker World of the Cell 7 th Edition Fig A-12

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Other useful fluorescent dyes
 DAPI (4',6-diamidino-2-phenylindole) and Hoechst stain bind to the minor groove
of DNA and emit blue light when exposed to UV light
 Phalloidin toxin from the death cap mushroom (Amanita phalloides) – binds to
filamentous actin (F-actin) and can be conjugated to different fluorophores

http://en.wikipedia.org/wiki/DAPI
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Moving mRNA vs moving protein
 Do you build houses and transport them to their street or do you take the plans
to a site where the builders and materials are?

 Makes lots of sense in certain cell types e.g. neurons

Axon length
<1mm to >1m Alberts 5th Edition Fig 11-28
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Cell migration
 Wound healing, movement of WBCs to infection sites, metastasis
 Requires rapid changes in the cytoskeleton
 The leading edge of the cell has lots of actin microfilaments
 These assemble/disassemble to push membrane forward
 New synthesis of actin protein is also required to help in the migration process

Red = Actin mRNA (detected by

FISH)
Green = Actin protein (detected by
phalloidin)
Blue = DNA in nucleus (detected
by DAPI)

http://flickr.com/photos/8237985@N03/2132037812/
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Features of a eukaryotic mRNA

Open Reading Frame (ORF)

mRNA 5’ UTR 3’ UTR
AAAA
AUG UAG
m7G cap UAA Poly(A) tail
Initiation
UGA
codon
Termination
codon

 Untranslated regions (UTRs) are involved in regulation

 Sequences in the 3’ UTR may be involved in localisation
 e.g. the b-actin mRNA contains a “zip code” which has an essential ACACCC
sequence
 The zip code forms a secondary structure via base-pairing within the 3’ UTR
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Zip code Binding Protein (ZBP1)

RRM RRM KH KH KH KH

 Contains 2x RRMs (RNA recognition motif) which has basic regions (positive
charge) and interacts with negative charge on RNA
 Also 4x KH domains (K-homology, first found in hnRNPK) which bind to single-
stranded RNA and DNA
 ZBP1 binds to nascent b-actin mRNA in nucleus as part of a larger mRNP complex
 These mRNPs are actively transported through the nuclear pore complex (NPC)
with the aid of Ran GTPase (see later)
Transport of b-actin mRNA to the leading edge
12

of a migrating cell

Shav-Tal and Singer, 2005 J. Cell Sci. 118: 4077-81

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Post-translational protein targeting
 Many proteins are made
in the cytoplasm and
THEN moved to where
they need to be

 They therefore require organelle-

specific targeting sequences

 Some of these can be identified

from the primary sequence (NLS,
NES)

 Some are more structural

 Amphiphilic helix
(mitochondria)
 Signal patch (lysosome)
Lodish 6th Edition, Fig 13-1
 Organelle-specific targeting: Entering the
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nucleus

SV40 large T antigen PKKKRKV

Nucleoplasmin KRPAAIKKAGQAKKKK
CBP80 RRRHSDENDGGQPHKRRK

 Requires a targeting sequence known as a Nuclear Localisation Signal (NLS)

which is NOT removed following transport
 Two kinds of NLS, may be anywhere in protein
 Basic e.g. SV40 large T antigen
 rich in lysine (K) and arginine (R)
 the exact sequence is not important instead it is the cluster of basic amino
acids
 can be bipartite (split into two parts)
 Non-basic e.g. hnRNPA1 is hydrophobic
 Organelle-specific targeting: Exiting the
15

nucleus

la Cour et al (2003) Nucleic Acids Research, 31, 393-396

 Requires Nuclear Export Signals (NESs)

 These can be anywhere in the sequence and are NOT removed following transport
 Rich in hydrophobic residues (leucine, L and isoleucine, I)
 Consensus L X2-3 (FILVM) X2-3 (LI) X (LI)
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A nuclear localisation signal in eIF4GI
Caspase-3 Caspase-3

eIF4GI
PABP NLS eIF4E eIF4A/eIF3 eIF4A Mnk

 Large protein that acts as scaffold in translation initiation

 Indirect immunofluorescence of HeLa cells shows some of the endogenous protein

is in the nucleus, most is in the cytoplasm

 We expressed fragments corresponding to known protease cleavage sites – N-

terminal part goes to nucleus (detected by indirect IF)
 N-terminal fragment sequence contains KRRRK

 Made fusion proteins with GFP - whole fragment or just a small fragment of
surrounding sequence (X5)
 Mutated KRRRK to AAAAA (charge goes from positive to neutral)
 Nuclear localisation was dependent on this sequence
 Getting into the nucleus –
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The nuclear pore complex

Nucleoporins

Lodish 7th Edition, Fig 13-33

 Ions, small metabolites and globular proteins 20-40 kDa move through the
nuclear pore complex (NPC) by passive diffusion
 Bigger proteins and mRNAs (in mRNPs) need to be transported through the pore
– central role of the GTPase Ran
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The nuclear import process

1. Nuclear import receptor:

heterodimer of importin-a
(recognises NLS in
“cargo”) and importin-b
(interacts with
nucleoporins in NPC)
2. Nucleoporins contain FG-
2 rich repeats (hydrophobic)
3. These help transport
(shuttle) the complex into
the nucleus

Lodish 7th Edition, Fig 13-36

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The nuclear import process

6 4. Nuclear Ran-GTP interacts

with importin, releasing
cargo
5. Ran-GTP:importin
complex exits to
cytoplasm
6. Ran-GAP stimulates Ran
2 7 to hydrolyse GTP
7. Importin is now free for
5 another round of import
8. Ran-GDP enters the
nucleus to be “recycled”
3 by Ran-GEF

4
Lodish 7th Edition, Fig 13-36
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The nuclear export pathway
 Again, the Ran GTPase is
central
 In this case the cargo is
included in a complex
4 with Ran-GTP and a
transport protein called an
exportin (e.g. Crm1)
 Exportins and importins
3 are similar in both
sequence and structure and
are part of a family called
2 karyopherins

1.
2.
1 3.
4.
5 5.
Lodish 6th Edition, Fig 13-37a
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Regulated nuclear export of Mnk1

Courtesy of
Dr Jo Cowan

 Mnk1 is a kinase with an N-terminal NLS and a C-terminal NES

 The top row shows myc-tagged Mnk1 protein detected by IF
 Leptomycin B (LMB) inhibits nuclear export of Mnk1
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Using Leptomycin B to prevent cancer
 Being able to keep proteins in their correct cellular compartments maybe important
therapeutically
 Leptomycin B (from Streptomyces) binds to and alkylates Exportin1 on a single Cys
residue, inhibiting the export activity
 LMB has been found to have anti-tumour activity in mouse models
 Lots of proteins with tumour suppressor activity need to be in the nucleus to carry
out their function
 The mislocalisation of tumour suppressor proteins is a common feature of tumours
 e.g. RUNX3 (a transcription factor) is commonly mislocalised to the cytoplasm in
breast and gastric tumours
 Blocking Exportin1-dependent nuclear export with LMB may help to keep such
proteins where they ought to be and prevent cancer progression
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Lecture 1 Conclusions
 Proteins in the cell need to be in the right place to perform their function
 This can be achieved by movement of the mRNA via sequences located in the 3’
UTR (Pre-translation)
 Movement of proteins Post-translation relies on targeting sequences
 The simplest of these signals are those that mark out a protein for nuclear import or
export
 All these movements can be visualised in cells by various methods
 Hybridisation of a fluorescent nucleotide probe to mRNA
 Binding of fluorescently labelled antibodies to proteins
 Expression of a protein of interest fused to a fluorescent protein

 Next time:
 Targeting to mitochondria
 Identifying localisation signals in novel open reading frames
 How proteins can be localised at the same time as they are being translated (Co-
translational transport)