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Prokaryotic protein targeting (secretion)

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The chaperone protein SecB binds to the nascent polypeptide chain to prevent premature folding which would make transport across the plasma membrane impossible. SecE and SecY are transmembrane components which form a pore in the membrane through which the still unfolded polypeptide is threaded. The translocation process is energy-dependent (ATP) and is driven by SecA. Once the protein has passed through the pore, the signal sequence is cleaved off by an extracellular, membrane-bound protease.

Eukaryotic Protein Targeting

Targeting in eukaryotes is necessarily more complex due to the multitude of internal compartments:

• nucleus
• mitochondria
• peroxisomes
• chloroplasts
• endoplasmic reticulum (ER)
• Golgi
• lysosomes
• secretory granules

There are two basic forms of targeting pathways:

• post-translational targeting:
• • nucleus
  • mitochondria
  • chloroplasts
  • peroxisomes

• co-translational targeting (secretory pathway):
• • ER
  • Golgi
  • lysosomes
  • plasma membrane
  • secreted proteins

In the absence of targeting signals, a protein will remain in the cytoplasm:

• translational machinery
• metabolic enzymes
• cytoskeletal proteins
• many signal transduction proteins

Nuclear targeting:

• Unusual since 2-way traffic:
• • in: proteins, DNA
  • • DNA & RNA polymerases
    • transcriptions factors
    • histones etc.
  • out: mRNA, tRNA, rRNA

• Proteins are not transported through the nuclear membrane but rather through a complex pore called the nuclear pore:
• • comprised of about 100 different proteins
  • proteins smaller than 20 kDa move by diffusion
  • proteins larger than 20 kDa move by selective transport (nuclear localization signal)
  • • cluster of 4-8 positively charged amino acids (example: PKKKRLV)
    • signal sequence binds to receptor on the pore called importin

Mitochondrial targeting:

• not well understood
• usually by post-translational targeting

Lysosomal targeting:

• Lysosomes are organelles that store enzymes which rapidly degrade other proteins and nucleic acids.
• A famous target sequence is "KDEL"
• Initial targeting via secretory pathway
• Final targeting occurs in the Golgi

The secretory pathway

ER targeting (secretory pathway)

• co-translational insertion of protein into or through ER membrane via attached ribosomes (rough ER):
• • signal sequence of 16-30 amino acids at N-terminus (hydrophobic)
  • emerging signal sequence of nascent protein on free ribosome binds to signal recognition particle (SRP) -- translation is arrested.
  • • SRPs consist of 6 proteins and one RNA molecule (7S RNA).
    • The SRP-signal sequence-mRNA-ribosome complex docks with receptor on ER membrane.
  • signal sequence crosses ER membrane.
  • translation continues with polypeptide chain being pulled into the ER lumen.

While in the ER, many proteins undergo the first stages of glycosylation. Most proteins then migrate inside vesicles from the ER and enter the cis face of the Golgi where further processing and final sorting occurs:

The Golgi Complex

The Golgi is responsible for further processing and final sorting of proteins. One example is the formation of primary and secondary lysosomes:

• Primary lysosomes bud from the trans face of the Golgi and subsequently
• • undergo exocytosis (A)
  • fuse with vesicles to digest their contents (B & C)
  • rupture, causing autolysis (D)

Overview of Trafficking

In order to keep a cell working it needs to remove:

• incorrectly synthesized proteins (with errors in amino acid sequence)
• damaged proteins (i.e. oxidative damage)
• cell-cycle specific proteins
• other signaling proteins which are no longer necessary

One mechanism of protein degradation is via lysosomes. Lysosomes are acidic vesicles that contain about 50 different enzymes involved in degradation:

• proteases (cathepsins): cleave peptide bonds
• phosphatases: remove covalently bound phosphates
• nucleases: cleave DNA/RNA
• lipases: cleave lipid molecules
• carbohydrate-cleaving enzymes: remove covalently bound sugars from glycoproteins

• Lysosomes often secrete their contents into the extracellular medium via exocytosis.
• Lysosomes can also target damaged organelles in a process called autophagy.
• Sometimes, lysosomes are triggered to rupture inside a cell, resulting in autolysis, also called apoptosis or programmed cell death.

Another major mechanism is via ubiquitin labeling of surplus proteins:

• Ubiquitin (a small 76-residue protein) is attached to the protein:
• • First, an activating enzyme attaches itself to the carboxy terminus of free ubiquitin in an ATP-dependent process.
  • Then, the activated ubiquitin is transferred onto a second enzyme which at the same time recognizes damaged proteins.
  • The activated ubiquitin is then covalently linked to lysine residues on the surface of the damaged protein.

• These ubiquitin-tagged proteins are now recognized by specific proteases in the cytosol which in turn cleave and degrade the tagged protein.

• These proteases are combined in a very large protein complex called the proteasome.

• The proteasome (20S) is comprised of 28 subunits and has a molecular weight of 700 kDa:

SUMMARY:

Protein Targeting and Sorting

Synthesis of all polypeptides encoded by nuclear genes begins in the cytosol.
The large and small ribosomal subunits associate with each other and with the 5 prime end of an mRNA molecule, forming a functional ribosome that starts making the polypeptide.
When the polypeptide is about 30 amino acids long, it enters one of two alternative pathways.
1) In cotranslational import, if the newly forming polypeptide is destined for any of the compartments of the endomembrane system, it becomes associated with the ER membrane and is transferred across the membrane into the lumen (cisternal space) of the ER as synthesis continues.
The completed polypeptide then either remains in the ER or is transported via various vesicles and the Golgi complex to another final destination.
Integral membrane proteins are inserted into the ER membrane as they are made, rather than into the lumen.

2) If the polypeptide is destined for the cytosol or for import into the nucleus, mitochondria, chloroplasts, or peroxisomes, its synthesis continues in the cytosol.
When the polypeptide is complete, it is released from the ribosome and either remains in the cytosol or is transported into the appropriate organelle by posttranslational import.

Polypeptide uptake by the nucleus occurs via the nuclear pores, using a mechanism different from that involved in posttranslational uptake by other organelles.

In cotranslational import, proteins to be targeted to the endoplasmic reticulum initially have an N-terminal peptide, the ER signal sequence, translated by a cytosolic ribosome.
The ER signal sequence is bound by a signal-recognition particle (SRP), a ribonucleoprotein complex composed of 6 peptides and a 300 nucleotide RNA molecule.
The SRP binds to the SRP receptor to dock the ribosome on the ER membrane.
When the SRP receptor binds GTP, the nascent polypeptide enters the pore.
The SRP is released with hydrolysis of the GTP.
The growing polypeptide translocates through a hydrophilic pore created by one or more membrane proteins called the translocon.
The most recent evidence suggests that the ribosome fits tightly across the cytoplasmic side of the pore and that the ER-lumen side is somehow closed off until the polypeptide is about 70 amino acids long.
When the polypepide is complete, the signal peptidase cleave the signal to release the protein into the ER lumen while retaining the signal peptide, for a time, in the membrane.
Afterwards the ribosome is released and the pore closes completely.

In the endoplasmic reticulum, folding of the newly-made proteins may also require molecular chaperones and other proteins involved in protein folding.
Bip (binding protein), a member of the Hsp70 chaperone family, briefly binds to and stabilizes hydrophobic regions of proteins (especially rich in Trp, Phe, Leu) allowing proper folding instead of aggregation with other inmature proteins.
Protein disulfide isomerase catalyses the formation and breakage of disulfide bonds between cysteine residues to produce a stable conformation.

There are two possible mechanisms for the insertion of integral membrane proteins having a single transmembrane segment.
1) Type I: Insertion of a polypeptide with both a terminal ER signal sequence and an internal stop-transfer sequence.
The terminal peptide is eventually cut off, leaving a transmembrane protein with its N-terminus in the ER lumen and its C-terminus in the cytosol.
2) Type II: Insertion of a polypeptide with only a single, internal start transfer sequence, which both starts polypeptide transfer and anchors itself permanently in the membrane.
The amino-carboxyl orientation of the completed protein depends on the orientation of the start-transfer sequence when it first inserts into the translocation apparatus.

Posttranslational import allows some polypeptides to enter organelles after protein synthesis.
Like cotranslational import into the ER, posttranslational import into a mitochondrion (and chloroplast) involves a signal sequence (called a transit sequence), a membrane receptor, pore-forming membrane proteins, and a peptidase.
Polypeptides being imported into the mitochondrion span both membranes at the same time.
This was demonstrated in a cell-free import system incubated on ice in which the polypeptides begin to penetrate the mitochondrion but then stall.
The transit sequence is cleaved by the transit peptidase present in the matrix, indicating that the N-terminus of the polypeptide is within the mitochondrion.
At the same time, most of the polypeptide molecule is can be attacked by exogenously added proteolytic enzymes on the outside of the mitochondrion.
Therefore, the polypeptide must span both membranes transiently during import at a contact site between the two membranes.

However, in the mitochondrion, the membrane receptor recognizes the signal sequence directly without the intervention of a cytosolic SRP.
Furthermore, chaperone proteins play several crucial roles in the mitochondrial process:
1) Chaperones keep the polypeptide partially unfolded after synthesis in the cytosol so that binding of the transit sequence and translocation can occur.
2) Chaperones drive the translocation itself by binding to and releasing from the polypeptide within the matrix, an ATP-requiring process and
3) Chaperones often help the polypeptide fold into its final conformation.

Polypeptides synthesized on cytosolic ribosomes but destined for either the intermembrane space or the inner membrane of the mitochondrion require two separate targeting sequences (both located at the N-terminus).
1) The polypeptide is directed to a contact (translocation) site on the mitochondrion by a positively charged or amphipathic transit sequence.
2) Cleavage of the transit sequence by a peptidase in the mitochondrial matrix uncovers a highly hydrophobic second signal sequence.
3) This second signal sequence causes the polypeptide to be inserted into the inner membrane in the same way that mitochondrially encoded polypeptides are targeted to this membrane.
4) The remainder of the polypeptide is then moved across the membrane into the intermembrane space (or into the inner membrane for integral inner membrane proteins).
5) Cleavage by a second peptidase can release the polypeptide into the intermembrane space leaving the signal sequence behind in the inner membrane.