Tema 12
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Tema 12 Localización y translocación proteica. CA García Sepúlveda MD PhD. Laboratorio de Genómica Viral y Humana Facultad de Medicina, Universidad Autónoma de San Luis Potosí.

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Ca garc a sep lveda md phd

Tema 12

Localización y translocación proteica

CA García Sepúlveda MD PhD

Laboratorio de Genómica Viral y HumanaFacultad de Medicina, Universidad Autónoma de San Luis Potosí


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Proteins can be classified into two general classes with regard to localization: those that are not associated with membranes; and those not-associated with membranes.

Each class can be subdivided further, depending on whether the protein associates with a particular structure in the cytosol or type of membrane.

Proteins can be localized co-translationally or post-translationally.

Tema 12. Localización y translocación proteicaIntroducción

Protein fate


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Proteins that are localized post-translationally are released into the cytosol after synthesis on free ribosomes.

Tema 12. Localización y translocación proteicaLocalización postraduccional

Protein fate


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Proteins that are localized post-translationally are released into the cytosol after synthesis on free ribosomes.

Some have signals for targeting to organelles such as the nucleus or mitochondria.

Tema 12. Localización y translocación proteicaLocalización postraduccional

Protein fate


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Proteins localized co-translationally associate with the ER membrane during synthesis, ribosomes are "membrane-bound".

Tema 12. Localización y translocación proteicaLocalización co-traduccional

Protein fate


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Proteins localized co-translationally associate with the ER membrane during synthesis, ribosomes are "membrane-bound".

The proteins pass into the ER along the Golgi and then through the plasma membrane, unless they have signals that cause retention at one of the steps on the pathway.

Tema 12. Localización y translocación proteicaLocalización co-traduccional

Protein fate


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Proteins localized co-translationally associate with the ER membrane during synthesis, ribosomes are "membrane-bound".

The proteins pass into the ER along the Golgi and then through the plasma membrane, unless they have signals that cause retention at one of the steps on the pathway.

They may also be directed to other organelles, such as endosomes or lysosomes.

Tema 12. Localización y translocación proteicaLocalización co-traduccional

Protein fate


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Cytosolic (or "soluble") proteins carry out functions in the cytosol.

The ribosomes on which these proteins are synthesized are sometimes called "free ribosomes".

The "default" for a protein released from "free" ribosomes is to remain in the cytosol.

To be targeted to a specific location requires an appropriate signal, typically a sequence motif that causes it to be assembled into a macromolecular structure or recognized by a transport system.

Tema 12. Localización y translocación proteicaProteinas citosolicas


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Some proteins remain free in the cytosol in quasi-soluble form; others associate with macromolecular cytosolic structures (filaments, microtubules, centrioles, etc).

This class also includes nuclear proteins (which pass into the nucleus through large aqueous pores).

Tema 12. Localización y translocación proteicaProteinas citosolicas


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aka: Endomembrane System

Series of membranous bodies, including ER, Golgi apparatus, endosomes and lysosomes.

Proteins of this system are inserted into the ER and then directed to their particular locations by the vessicletransport system.

Tema 12. Localización y translocación proteicaSistema reticuloendotelial

  • Proteins that are secreted from the cell are transported to and through the plasma membrane to the exterior.


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There are three major subdivisions of the endomembrane system

the secretory pathway

the lysosomal pathway and

the endocytotic pathway

Tema 12. Localización y translocación proteicaSistema reticuloendotelial


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There are three major subdivisions of the endomembrane system

the secretory pathway

the lysosomal pathway and

the endocytotic pathway

Tema 12. Localización y translocación proteicaSistema reticuloendotelial


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There are three major subdivisions of the endomembrane system

the secretory pathway

the lysosomal pathway and

the endocytotic pathway

Tema 12. Localización y translocación proteicaSistema reticuloendotelial


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Once proteins enter the endoplasmic reticulum they never return to the cytosol compartment.

They are carried by vesicle transport to the other compartments of the system.

This flow of vesicles is highly regulated.

Tema 12. Localización y translocación proteicaSistema reticuloendotelial


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Consists of compartments:

Endoplasmic Reticulum

Golgi apparatus

Lysosomes

Endosomes and

Secretory Vesicles.

Tema 12. Localización y translocación proteicaSistema reticuloendotelial


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Compartments involved in the processing of proteins for:

export from the cell

for lysosomes (destruction)

for proteins entering the cell from the cell surface.

Tema 12. Localización y translocación proteicaSistema reticuloendotelial


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Compartments involved in the processing of proteins for:

export from the cell

for lysosomes (destruction)

for proteins entering the cell from the cell surface.

Tema 12. Localización y translocación proteicaSistema reticuloendotelial


Ca garc a sep lveda md phd

Compartments involved in the processing of proteins for:

export from the cell

for lysosomes (destruction)

for proteins entering the cell from the cell surface.

Tema 12. Localización y translocación proteicaSistema reticuloendotelial


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The process of inserting into or passing through a membrane is called protein translocation.

Protein translocation is driven by signals intrinsic to the proteins themselves.

Tema 12. Localización y translocación proteicaLocalización proteica


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Nuclear localization signals (short sequences within proteins) enable the proteins to pass through nuclear pores.

One type of signal that determines transport to the peroxisome is a very short C-terminal sequence.

Tema 12. Localización y translocación proteicaSeñales de localización

  • Insulin signal peptide →


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Tema 12. Localización y translocación proteicaSeñales de localización a RER

Synthesis of all proteins begins in the cytosol compartment.

For proteins entering the secretory or lysosomal pathways, the first step is targeting to the ER.


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Tema 12. Localización y translocación proteicaSeñales de localización a RER

This targeting relies on a signal encoded in the N terminal portion of the protein.


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Tema 12. Localización y translocación proteicaSeñales de localización a RER

The signal is recognized by a Signal Recognition Particle (SRP).


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Tema 12. Localización y translocación proteicaSeñales de localización a RER

The SRP enables the ribosome to dock to the corresponding translocator protein (translocon).


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Tema 12. Localización y translocación proteicaSeñales de localización a RER

The SRP enables the ribosome to dock to the corresponding translocator protein (translocon).


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Tema 12. Localización y translocación proteicaSeñales de localización a RER

Signal sequence provides the same traffikcing pattern for completely distinct proteins...


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Tema 12. Localización y translocación proteicaSeñales de localización a RER

Nascent polypeptide is inyected into ER and the signal sequence is cleaved by a Signal Peptidase.


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Tema 12. Localización y translocación proteicaSeñales de localización a RER

Protein synthesis continues to completion until the ribosome is undocked & dissociated.


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Tema 12. Localización y translocación proteicaSeñales de localización a RER

This is a prime example of a co-translationally localized protein...now on to explore post-translational localization...


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Tema 12. Localización y translocación proteicaSeñales de localización a RER

What about proteins synthesized in the cytosol that are incorporated to the ER ?


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Tema 12. Localización y translocación proteicaSeñales de localización a RER

What about proteins synthesized in the cytosol that are incorporated to the ER ?

The peptide moves through the translocon into the lumen of the ER.

The signal peptide remains attached to the membrane.

Signal peptide is cleaved off by a signal peptidase.Leaving the protein free in the lumen of the ER.


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Tema 12. Localización y translocación proteicaProteinas de membrana

And what about proteins that become an INTEGRAL PART OF THE ER MEMBRANE ?


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Tema 12. Localización y translocación proteicaProteinas de membrana

As membrane proteins are being translated, they are translocated into the ER until a hydrophobic domain is encountered. Alpha helices serve as a 'stop transfer' signal and leaves the protein inserted in the ER membrane.


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Tema 12. Localización y translocación proteicaProteinas de membrana

The orientation of a protein in the membrane is established when it is first inserted into the membrane.

This orientation persists all of the way to its final destination.

That is, the cytosolic side of membrane remains on the cytosolic side throughout all processes.


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Tema 12. Localización y translocación proteicaProteinas de membrana

Classification based on the way the integral proteins are inserted into the membrane and on the times they pass through it.


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Tema 12. Localización y translocación proteicaProteinas de membrana

Type I : Single pass, N-terminus in extracellular or luminal space.

Leader sequence in N-terminus

Leader sequence is cleaved inside the ER lumen.


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Tema 12. Localización y translocación proteicaProteinas de membrana

Type II : Single pass, C-terminus in extracellular or luminal space.

Leader sequence absent but protein introduced C-terminus first.


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Tema 12. Localización y translocación proteicaProteinas de membrana

Type III : Polypeptide crosses the lipid bilayer multiple times (α-helix rich)

Even (2,4,6) number of hydrophobic domains N- and C- on same side

Odd (1,3,5) number of hydrophobic domains N- and C- on different sides


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Tema 12. Localización y translocación proteicaProteinas de membrana

Lipid chain-anchored membrane proteins and GPI-anchored membrane proteins : Associated with the bilayer only by means of one or more covalently attached fatty acid chains.The latter is bound to the membrane by a glycosylphosphatidylinositol (GPI) anchor.


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Tema 12. Localización y translocación proteicaProteinas de membrana

Luminal side becomes extracellular side for some proteins.


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Mitochondrial and chloroplast proteins are synthesized on "free" ribosomes.

They associate with the organelle membranes by means of N-terminal sequences of ~25 amino acids that are recognized by receptors on the organelle envelope.

Tema 12. Localización y translocación proteicaProteinas mitocondriales

  • Because this process takes place after synthesis of the protein has been completed, it is called post-translational translocation.


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Same as for ER.

Requires specific translocons and SRP.

As endosymbionts have two membranes, two different types of translocons are needed

TOM

TIM

Tema 12. Localización y translocación proteicaProteinas mitocondriales

  • Incorporated proteins can be integrated into membranes as happens for ER proteins.


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The "default pathway" takes a protein through the ER, into the Golgi, and on to the plasma membrane.

Tema 12. Localización y translocación proteicaProteinas secretadas


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A polarized thyroid epithelial cell synthesizing soluble proteins:

Polypeptides generated by RER membrane-bound polysomes, enter the lumen of RER.

Proteins undergo core glycosylation and by interacting with chaperones acquire their conformation.

Tema 12. Localización y translocación proteicaTráfico en células polarizadas

  • Proteins are then transported to the Golgi apparatus, where terminal glycosylation and other post-translational reactions take place.


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In the Trans-Golgi network (TGN), mature proteins undergo sorting processes and are packed into transport vesicles.

The vesicles carry soluble proteins (inside the vesicle) and membrane proteins (as integral vesicle membrane protein).

Tema 12. Localización y translocación proteicaTráfico en células polarizadas


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Proteins that reside in the ER possess a C-terminal tetrapeptide KDEL (Lys-Asp-Glu-Leu) which signals their returnto the ER from the Golgi.

Tema 12. Localización y translocación proteicaTráfico en células polarizadas

COPI is a protein that coats vesicles that transports proteins from the cis end of the Golgi complex to the RER. This type of transport is termed retrograde transport.


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Tema 12. Localización y translocación proteicaJerarquias de secuencias lider

  • Mitochondria synthesize only ~10organelle proteins; chloroplasts ~50.

  • The majority of organelle proteins are synthesized in the cytosol by free ribosomes. They must then be imported into the organelle.

  • Post-translational membrane insertion depends on LEADER SEQUENCES.

  • Leaders for mitochondria/chloroplasts are usually hydrophilic, consisting of uncharged amino acids interrupted by basic amino acids, and lacking acidic amino acids.


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Tema 12. Localización y translocación proteicaJerarquias de secuencias lider

  • The leader sequence contains all the information needed to localize a protein.

  • The leader sequence and the transported protein represent domains that fold independently to be recognized by receptors on the organelle envelope.

  • The attached polypeptide sequence plays no part in recognition of the envelope.

  • Complexity of endosymbiont proteins =

    • outer membrane

    • the intermembrane space

    • the inner membrane

    • the matrix.


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Tema 12. Localización y translocación proteicaJerarquias de secuencias lider

  • A hierarchy of leader signals tells each protein where to localize.

  • The default endosymbiont pathway for protein localization takes a protein completely into the matrix.

  • This requires two signals (in the leader):

    • Organelle recognition & outer membrane passage (first part of the leader sequence).

    • Inner Membrane recognition & passage (second part).

  • Proteins that need to be held in intermembranespace or as integral inner membrane proteins require even more signals.


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Tema 12. Localización y translocación proteicaJerarquias de secuencias lider

  • This requires two signals (in the leader):

    • Organelle recognition & outer membrane passage (first part of the leader sequence).

    • Inner Membrane recognition & passage (second part).

Many uncharged amino acids

Basic amino acids


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Tema 12. Localización y translocación proteicaTranslocones

  • There is a basic problem in passing a (largely) hydrophilic protein through a hydrophobic membrane.

  • The energetics of the interaction are highly unfavorable.

  • Translocating proteins move through an aqueous channel (translocon), interacting with the resident (integral) proteins rather than with the lipid bilayer.


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Tema 12. Localización y translocación proteicaTranslocones

  • When the signal sequence enters the translocon, the ribosome attaches, forming a seal so that the pore is not exposed to the cytosol.

  • Ribosome is bound by the interaction of the Signal Recognition Particle (SRP) and the SRP-receptor.


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Tema 12. Localización y translocación proteicaTranslocones

  • Sec61 Complex is the major component of the translocon:

    • Sec61α

    • Sec61β

    • Sec61γ

  • Forms cylindrical oligomers (each of 3 to 4 heterotrimers) with a diameter of ~8.5nm and a central pore of ~2 nm.


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Tema 12. Localización y translocación proteicaTranslocones

  • A more complex translocon is required when a protein is inserted into a membrane post-translationally.

    • Sec61 complex

    • Four other Sec proteins

    • Chaperone BiP (a member of the Hsp70 class)

    • Supply of ATP

  • BiP prevents protein backslash due to Brownian Motion.


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Tema 12. Localización y translocación proteicaComplejo del poro nuclear

  • The nucleus is segregated from the cytoplasm by an envelope consisting of two membranes.

  • The outer membrane is continuous with the ER in the cytosol.

  • The two membranes come into contact at openings called nuclearpore complexes (~3000 per cell).

Pore provides a water-soluble channel between nucleus and cytoplasm.

Nucleus and cytosol have the same ionic milieu !


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Tema 12. Localización y translocación proteicaComplejo del poro nuclear

  • Nuclear pores are used for both import and export of material.

  • Proteins are synthesized in the cytosol so any protein required in the nucleus must be transported there.

  • Since all RNA is synthesized in the nucleus, the entire cytoplasmic complement of RNA (mRNA, rRNA, tRNA, and other small RNAs) must be exported from the nucleus.


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Tema 12. Localización y translocación proteicaComplejo del poro nuclear

  • The entire pore complex has a diameter of about 120 nm.

  • Pore diameter is 50 nm wide and its "depth" is about 200 nm.

  • Mammalian is 120 MDalton and contains approximately 30 different protein components.


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Tema 12. Localización y translocación proteicaComplejo del poro nuclear

  • Molecules of <5 kD that are injected into the cytoplasm appear virtually instantaneously in the nucleus.

    • Freely permeable to ions, nucleotides and other small molecules.

  • Proteins between 5-50 kD diffuse at a rate that is inversely related to their size.

    • Presumably determined by random contacts with the pore.

    • It takes a few hours for the levels of an injected protein to equilibrate between cytoplasm and nucleus.

    • Small proteins can enter the nucleus by passive diffusion (but they may also be actively transported).

  • Proteins >50 kD in size do not enter the nucleus by passive diffusion.

    • Active transport required for their passage


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Tema 12. Localización y translocación proteicaComplejo del poro nuclear

  • For a protein to pass through a NPC it must have a special signal sequence.

  • The most common motif responsible for import into the nucleus is the Nuclear Localization Signal (NLS).

  • Its presence is necessary and sufficient to sponsor import into the nucleus.

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

  • There is no apparent conservation of sequence of NLS signals

    • short, rather basic stretch of amino acids.

    • Proline residue usually breaks α-helix upstream of basic residues.

    • Hydrophobic residues are rare.


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Tema 12. Localización y translocación proteicaComplejo del poro nuclear


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