Protein targeting: insertion into the ER membrane

Protein targeting:

insertion into the ER membrane

promoter coding region

messenger RNA

messenger RNA

Nascent protein

Gene transcription

by RNA-polymerase

Gene translation

by ribosomes

Protein folding

and transport

(Genomic DNA strand)

Endoplasmic reticulum

Essential chapter in Molecular Biology of the Cell,

Alberts et al., 4rth or 5th edition

Chapter 12

Intracellular compartments and protein sorting

Page 695-749

Protein synthesis, soluble or

membrane spanning?

Two synthesis routes for nuclear encoded genes:

1) Cytosol (soluble ribosomes)

2) Endoplasmic reticulum surface (membrane

bound ribosomes)

Both synthesis routes can lead to soluble as well

as membrane proteins

How are membrane proteins synthesized?

Antibody coding regions contain N-terminal

signal peptides

After translocation,

the signal peptides are cleaved

“Mature” poly-peptides

“Nascent” polypeptides

Assembly to tetramers

Soluble protein after translocation

Lumen

or extracellular

matrix

Cytosol

But some antibodies are membrane spanning

Membrane

bound IgM:

Relevant to our

Immune system

ER-, Golgi-,

vesicle- lumen,

or extracellular

matrix

Cytosol

Membrane proteins

can be classified into different groups

Phospholipid bilayer

ER lumen (oxidising)

Topologically like the

outside of the cell

Cytosol (reducing)

N-terminus (the beginning of the protein)

C-terminus (the end of the protein)

Type 1

Membrane protein

Membrane proteins

can be classified into different groups

Phospholipid bilayer

ER lumen (oxidising)

Topologically like the

outside of the cell

Cytosol (reducing)

C-terminus

N-terminus

Type 2

Membrane protein

Some proteins span the membrane many times

Phospholipid bilayerMultiple membrane spanning

protein

Some proteins span the membrane many times

A range of topologies is possible

NC

NC

N

C

C

N

The various transmembrane domains can be predicted

from the primary structure deduced from the coding region

The difference between membrane spanning proteins and membrane proteins

Phospholipid bilayer

Membrane spanning proteins

ER lumen (oxidising)

Topologically like the

outside of the cell

Cytosol (reducing)

Peripheral

membrane protein

Peripheral

Membrane

protein

How are antibodies inserted into membrane?

The heavy chain is

membrane spanning

The light chain is soluble,

but is membrane associated

via its interaction with the

heavy chain.

ER-, Golgi-,

vesicle- lumen,

or extracellular

matrix

Cytosol

Transmembrane domains are not cleaved and

remain part of the mature peptide

After translocation,

the signal peptides are cleaved

“Mature” poly-peptides

“Nascent” polypeptides

Assembly to tetramers

tethered to the membrane

TM domain

Cytosolic tail

Translocation of type I membrane spanning

proteins starts the same way as for soluble proteins

Portion coding for the

transmembrane domain

Cleavage of the signal peptide

Translocation of type I membrane spanning

continues like this until the transmembrane domain

enters the translocation pore

Portion coding for the

transmembrane domain

When the transmembrane domain reaches the

translocation pore, it stops translocation, hence the

name “stop-transfer” for the TM domain

Portion coding for the

transmembrane domain

Protein synthesis completes, the transmembrane

domain remains associated with the translocation

pore and the cytosolic tail remains in the cytosol

Lateral diffusion from the translocation pore into

the membrane and further folding

Phospholipid bilayer

ER lumen (oxidising)

Topologically like the

outside of the cell

Cytosol (reducing)

N-terminus (the beginning of the protein)

C-terminus (the end of the protein)

Type 1

Membrane protein

SP TM

Coding region of a type I membrane protein

cytosoliclumenal

Phospholipid bilayer

ER lumen (oxidising)

Topologically like the

outside of the cell

Cytosol (reducing)

Smaller N-terminal domain

Bigger C-terminal domain

Type 1

Membrane protein

SP TM

Coding region of another type I membrane protein

cytosoliclumenal

Membrane proteins

can be classified into different groups

Phospholipid bilayer

ER lumen (oxidising)

Topologically like the

outside of the cell

Cytosol (reducing)

N-terminus

C-terminus

Type 2

C-terminus

N-terminus

Type 1

Type II membrane proteins have no signal peptide

Phospholipid bilayer

ER lumen (oxidising)

Topologically like the

outside of the cell

Cytosol (reducing)

C-terminus

N-terminus

Type 2

Membrane protein:

(No signal peptide!)

TM

Type II membrane spanning proteins are often

post-translationally translocated

Portion coding for the

transmembrane domain

Less clearly

established

N-terminus

Type II membrane spanning proteins are often

post-translationally translocated

Less clearly

established

C-terminus

N-terminusSRP-dependent or independent

(competition assays in vitro)

Some type II membrane proteins are called

tail-anchored”

Phospholipid bilayer

ER lumen (oxidising)

Topologically like the

outside of the cell

Cytosol (reducing)

C-terminus

N-terminus

Extreme case of Type 2

Tail anchored

(No signal peptide!)

TM

Tail anchored molecules are always

post-translationally translocated

Novel SRP-independent route,

first published in 2007, translocation pore unknown

Transmembrane domain-

encoding

The protein leaves the

ribosome before SRP

could possibly bind

Post-translational modification

Folding and assembly into homo or hetero multimers

Proteolytic cleavage of portions

Glycosylation

Myristoylation,

Prenylation,

Phosphorylation

CN C C

O

G

HH

O

Protein anchored to membrane by

Fatty acid chain linked to N-terminal

Glycine (G: the side chain is H)

Amide

linkage

Post-translational modification

CN C C

O

H

HH

O

Protein anchored to membrane by

Fatty acid chain linked to N-terminal

glycine

Other examples of membrane anchoring include

GPI anchoring, prenylation (farnesyl or geranylgeranyl group)

or palmitylation (linking to palmitic acid) and involve other

amino acids (i.e. cysteine) and often the protein C-terminus.

Amide

linkage

NC

Myristic

acid

Membrane linkage via C-terminal prenylation

C

CH2

S

CH2

C O CH3

H O

N

CCysteine

Methylated

C-terminus

Where do we find membranes?

NE

RG

Lytic vacuole

Storage vacuole

Secretion

Mitochondria

Chloroplasts

Peroxisomes

Signals direct proteins to

Organelles

• With the exception of a few plastid and

mitochondrial proteins the proteins

destined for organelles are made in the

cytosol.

• To be incorporated into the right

organelle they need a signal within the

protein and a receptor which specifically

recognises the signal

Three mechanisms of transport

• Gated transporte.g. between nucleus and cytoplasm, mediated by

nuclear pore complexes

• Transmembrane transporte.g. across the membrane of the ER, mitochondria,

plastid or peroxisome

• Vesicular transporte.g. between organelles of the endomembrane system

(the secretory pathway)

Two main-stream protein targeting groups

Continued synthesis

on rough ER

Translocation

at the ER

Further transport via

vesicle budding and fusion

ER export via vesicles to

reach the Golgi apparatus

Golgi export in vesicles

destined to various places

Cytosolic

Nuclear

mitochondria

chloroplasts

peroxisomes

Nuclear export of mRNA

Translation of mRNA in the cytosol

1) Synthesis in cytosol or 2) SRP arrest

Transport to the nucleus

Proteins are not synthesized

in the nucleus

To act in the nucleus, they

have to be imported from the

cytosol

Examples:

Polymerases

Transcription factors

Ribosomal proteins

Typically 5 mm

in diameter

Possible Evolutionary Origin of the Nuclear

Membrane

Note the ER and nuclear membrane are continuous

More than 50 different proteins

called nucleoporins make up the

nuclear pore

Water filled pore

Scanning EM of the nucleus side of the nuclear

envelope

The nuclear pore complex from

the cytoplasmic side.

The membrane has been removed

with detergent.

Section of

nuclear

membrane,

showing that

the inner and

outer envelopes

are continuous.

•Molecules < 5,000 MW are freely permeable (e.g ATP, cofactors, small metabolites)

•Molecules of 17,000 MW equilibrate slowly (e.g. small proteins)

•Molecules of 60,000 (e.g.larger proteins) require active transport

9 nm

This equates to a channel size of 9 nm.

However much larger proteins and ribosome subunits have to move between

nucleus and cytoplasm. How is this achieved?

Nuclear targeting signals

• Usually a stretch of basic (i.e. amino acids

that are positively charged at cellular pH)

• The exact sequence and the location in

the protein are not usually important

T- antigen is a viral protein that works in the

Nucleus.

It is identified here by a fluorescent antibody.

When the second lysine (basic amino acid) is

changed to a threonine, the protein can

no longer accumulate in the nucleus. This shows

the lysine is NECESSARY for the function of

the signal.

If the sequence PPKKKRKV is attached

to a cytosolic protein it can confer nuclear

localisation. It is therefore SUFFICIENT to

function as a NLS.

Nuclear import and export is receptor-mediated

Mitochondria

The “powerhouse” of the cell

Outer membrane

Inner membrane: highly folded, 5-fold the surface of the outer

membrane, contains respiratory chain molecules,

ATP synthetase, and transport proteins.

Inter membrane space: similar to cytoplasm and

contains enzymes that use ATP

Matrix: Mitochondrial genome,

Protein synthesis machinery

Variety of enzymes

0.5 –1 mm

In diameter

(like bacteria)

4 different places for proteins to go

within the mitochondria

matrix Outer membrane Inner membrane Inter membrane

space

Example: Transport into the matrix

•Mitochondrial presequences do not have a

specific primary amino acid sequence

•They are enriched in BASIC (red)

HYDROPHOBIC (yellow) and POLAR usually

hydroxylated amino acids (blue).

•When folded into an alpha helix the basic and

polar residues lie predominantly on one face

of the helix and the hydrophobic residues on the

other.

•This feature is recognised by the presequence

receptor proteins

Features of mitochondrial presequences

TOM and TIM complexes

interact to form a ‘contact site’

at which proteins transported

into the matrix can cross both

membranes

Recognise

the presequence

Transport into the Matrix

Targeting to chloroplasts or plastids

Figure 1-44 from Biochemistry and Molecular Biology of Plants Buchanan et al.,

Chloroplasts are just one member of a family

of organelles called plastids

Development of chloroplasts from etioplasts

Is light dependent

Increasing time in light

Genes encoding photosynthetic proteins and enzymes are expressed,

the proteins translated and imported into the organelle.

The colourless chlorophyll precursor protochlorophyllide is converted to

Chlorophyll.

The prolamellar body (PB) contains very high amounts of lipids that assemble

with the newly synthesised membrane proteins to form the thylakoids

Fig 4.4 Biochemistry and Molecular Biology of Plants Buchanan et al.,

Protein import has been

studied mainly in chloroplasts

because it is easy to get

enough material

Import is post translational

Signals are often composites

and transport can occur in

multiple steps

Transport to the thylakoid space, several steps:

Outer membrane

Inner membrane

Chloroplast

Inter membrane space

Stroma

Thylakoid space

Chloroplast

transit peptide

Thylakoid

signal sequence

Mature

peptide

Chloroplast targeting signals

•No conserved primary amino acid sequence

•Forms a random coil in aqueous solution

Stromal transit peptide when fused to a passenger protein

directs transport to the stroma.

When deleted protein stays in cytoplasm

Therefore necessary and sufficient

Experimental evidence for a role in transport:

fusion and deletion

Protein import machinery of chloroplasts

TOC

TIC

cytosol

Outer envelope

Inner envelope

Stroma

Chaperones

Chaperones

TOC = Translocon of the Outer Chloroplast envelope

TIC =Translocon of the Inner Chloroplast envelope

Mechanism of import I

Ribosome-nascent

chain

Chloroplast protein

Bound to cytosolic

chaperone

TOC

ATP

GTP

Protein handed

Over to TOC machinery

Two of the proteins of the TOC complex are GTPases whose receptor activity

Is regulated by GTP binding and hydrolysis a bit like SRP and the SRP receptor

in ER targeting.

Mechanism of Import II

TOC

TIC

cytosol

Outer envelope

Inner envelope

Stroma

Chaperones

ATP

ADP

Chaperones

ATP

ADP

Stromal

peptidase

ATP hydrolysis by chaperones in the intermebrane space and stroma traps/pulls

the protein across the membrane.

Entry into the thylakoid space is very similar to translocation into the endoplasmic reticulum

or secretion across the plasma membrane in bacteria

Chloroplast

transit peptide

Thylakoid

signal sequence

Mature

peptide

In the thylakoid

In the stroma

In the cytosol

bacterium

‘Complex’ plastids in protists

• Some photosynthetic eukaryotes (e.g. dinoflagellates,

euglena) arose as a result of a secondary

endosymbiosis when a photosynthetic eukaryote (which

already had a chloroplast) was engulfed by another

eukaryotic cell.

• As a result their plastids are surrounded by three or

more membranes and are called complex plastids

• Proteins targeted to the complex plastids are first

targeted to the ER and then possibly via vesicles to the

plastid.

Peroxysomes have a main function in

• Breaking down fats

In mammals peroxisomes and mitochondria

co-operate to break down fatty acids.

Peroxisome enzyme systems shorten long

chain fatty acids, which are then exported

to mitochondria to finish the job.

Peroxisomes also deal with branched

Fatty acids like phytanic acid that are important

components of the diet

In plants and yeasts peroxisomes are the ONLY place in the cell where fatty acids are

degraded

Targeting to peroxisomes

• Unlike mitochondria and chloroplasts

peroxisomes have just a single membrane

and no DNA

• Like mitochondria and chloroplasts they

import proteins post translationally

• Unlike mitochondria and chloroplasts they

import proteins that are FOLDED

Peroxisomal proteins are targeted in two ways

PTS1: Soluble proteins use C-terminal SKL signals and

variants (i.e. SRL)

PTS2: membrane proteins are possibly targeted from the ER,

but this is currently under debate

SKL

H2N-(R/K)X6(Q/H)(L/A)

The signals are recognised by specific receptorsMuch less is known about the translocation mechanism