Transkripcija - eng.

To be transported through the nuclear pore, a protein must

have a special signal in its sequence. The most common

motif responsible for import into the nucleus is called the nuclear

localization signal (NLS). Its presence in a cytosolic protein

is necessary and sufficient to sponsor import into the

nucleus. Mutation of the signal can prevent the protein from entering

the nucleus.

The summary of nuclear localization signals in Figure 8.58

shows that there is no apparent conservation of sequence of NLS

signals; perhaps the shape of the region or its basicity are the

important features. Many NLS sequences take the form of a

short, rather basic stretch of amino acids. Often there is a proline

residue to break α-helix formation upstream of the basic

residues. Hydrophobic residues are rare. Some NLS signals are bipartite

and require two separate short clusters. Competition studies suggest

that NLS sequences are interchangeable, suggesting that they are all

recognized by the same import system.

Many exported proteins have a common type of signal that is necessary

and sufficient for the protein to move from the nucleus to the cytosol.

It is called an NES (nuclear export signal), and typically consists

of an ~10 amino acid sequence. The only common feature in the NES

sequences in different proteins is a pattern of conserved leucines.

A protein may have both an NLS and an NES, the former used for its

import into the nucleus, and the latter for its export. They may function

constitutively, or their use can be regulated, for example, by association

with other proteins that obscure or expose the relevant sequences.

8.28 Transport receptors carry cargo proteins

through the pore

Key Concepts

• Transport receptors have the dual properties of recognizing NLS or

NES sequences and binding to the nuclear pore.

• Exportins transport substrates from nucleus to cytoplasm;

importins transport substrates from cytoplasm to nucleus.

• Exportins and importins interact with nucleoporins in the pore.

Figure 8.58 Nuclear localization signals

have basic residues.

The basic principle of import and export is illustrated in Figure

8.59. A carrier protein (or transport receptor) takes the substrate

through the pore. The transport receptor must then be returned across

the membrane to function in another cycle. The transport receptors are

classified according to the direction in which they transport the cargo.

Importins bind the cargo in the cytoplasm and release it in the nucleus.

Exportins bind the cargo in the nucleus and release it in the cytoplasm.

There are multiple pathways for import and export. Transport for all

of the substrates for any particular pathway can be inhibited by saturating

that pathway with one of its substrates. Figure 8.60 summarizes the

independent pathways. At least two different types of pathways exist for

import of proteins; and each class of RNA is exported by a different

system. Each transport receptor recognizes a particular type of sequence

in its substrate, thus defining the specificity of the system.

The first handle on the process of import was provided by systems

for transport that depend upon the presence of an NLS in the substrate

protein. An in vitro assay for nuclear pore import has been developed by

using permeabilized cells. When cells are treated with digitonin, the

Transport receptors carry cargo proteins through the pore SECTION 8.28 227

Figure 8.61 The assay for nuclear pore

function uses permeabilized cells.

22 CHAPTER 8 Protein localization

plasma membrane becomes permeable, but the nuclear

envelope remains intact. Labeled proteins can be imported

into the nucleus in a process that is dependent upon

the provision of cytosolic components. Figure 8.61

shows how this system has been used to characterize the

transport process.

Transport can be divided into two stages: docking,

which consists of binding to the pore; and translocation,

which consists of movement through it. In the absence of

ATP, proteins containing a nuclear import signal can bind

at the pore, but they remain at the cytoplasmic face. A cytosolic fraction

is needed for binding. When ATP is provided, proteins can be translocated

through the pore. A different cytosolic fraction is needed to support

translocation. The need for cytosolic fractions at both stages reinforces

the view of the pore as a structure that provides the framework for transport,

but that does not provide all of the necessary facilities for handling

the substrates.

Figure 8.62 summarizes the functions of the components involved

in nuclear import. The transport receptor is the key intermediate in the

docking reaction. It can bind to both the nuclear pore and the cargo protein.

Some transport receptors are single proteins, such as transportin or

importin^3, which undertake both binding reactions. Others are

dimers in which one subunit binds to the pore, and the other is an adaptor

that binds to the cargo protein. In the best characterized case, importin-

o^ has a β subunit that binds to the nuclear pore. The a subunit

binds proteins that have an NLS sequence. The single protein receptors

transportin and importin^3 are related to the β subunit of importin-o^.

Translocation through the pore is inhibited by wheat germ agglutinin,

a lectin (glycoprotein). The component proteins of the pore that

bind to lectins were originally called nucleoporins. Note that nucleoporin

has since come to be used to mean any component of the nuclear

pore complex. The lectin-binding components are localized at or near

the region of the central pore, and appear to be located on both sides of

the nuclear envelope. When they are removed, pore complexes remain

normal in appearance, but can no longer function to transport large material.

Material smaller than the pore size continues to be able to move

through by diffusion. When the lectin-binding proteins are added back,

they restore full activity to the deficient pores. This suggests that they

are needed for active transport of material larger than the resting diameter.

Some of these proteins have some simple peptide repeating motifs

(GKFG, FG, FXFG), and it is probably these motifs that bind the importing

carrier proteins. They are called FG-nucleoporins.