BIOMG 1350 Lecture 10 - Protein Sorting

N-linked glycosylation and the Attachement of an Oligosaccharide to a Polypeptide in the ER

• During synthesis of proteins on the ER, whenever the sequence Asn-X-Ser/Thr comes through the translocon, a large oligosaccharide is transferred from a lipid-oligosaccharide donor to the asparagine side chain. This is called N-linked glycosylation. It never occurs on proteins in the cytosol.

• Many proteins are glycosylated, or sugars are added, on certain asparagine side chains in the ER. The protein must have a sequence where the complicated sugar tree can be attached.

• This is also called N linked because the sugar chain is attached to the nitrogen on the asparagine side chain. This process of a sugar being attached to the polypeptide only ever occurs in the ER lumen.

Necessity of Cellular Protein Sorting

• Most proteins remain in the cytosol though occasionally need to be transported into various organelles within the cell in order for the cellular functions to occur.

• Proteins are either produced within the rough ER or in the free floating ribosomes in the cytosol

• Proteins are sorted through several mechanics and are transported into organelles

Four Basic Pathways for Proteins in the Cell

Four basic pathways for proteins to travel on in the cell.

• (1) Cytosolic proteins are simply synthesized in the cytosol.

• (2) Proteins destined for mitochondria and chloroplasts are synthesized in the cytosol and transported across membranes into these organelles.

• (3) Proteins destined for the nucleus are also made in the cytosol and then transported in through nuclear pores.

• (4) Proteins destined for the ER, Golgi, endosomes, plasma membrane and to be secreted, are synthesized by the ‘secretory pathway’, and their synthesis occurs on the endoplasmic reticulum.

Protein Signal Sequences

• Signal sequences are necessary to direct a protein to its destination

• Surprisingly, there is not one precise sequence for each compartment, but common features for each compartment. For example, the signal sequence for targeting to the nucleus usually has many basic residues, whereas the ER signal sequence has many hydrophobic residues.

• Many of these signal sequences are located near the N-terminal part of the protein amino acid chain

• A cytosolic protein does not contain any signal sequence as it is created within the space it needs to be within and doesn’t need to be transported

What makes the Signal Sequence is Necessary and Sufficient?

• A signal sequence is a stretch of amino acid residues that is both necessary and sufficient to target a protein. For example, if molecular techniques are used to fuse a nuclear targeting sequence to a cytosolic protein, it will be driven into the nucleus.

• Proteins whose use is necessary in the ER, must contain a signal sequence so it can be properly transported from the cytosol, where it has been produced, into the ER

• If you add a signal sequence for entry into the ER to a protein that is normally just within the cytosol, then that protein will be transported into the ER. If you take away a signal sequence from a protein that normally targets the ER, then that protein will no longer go to the ER.

Signal Sequences, GFP/RFP, and the Illumination of specific Organelles

We can use signal sequences to target fluorescent proteins GFP/RFP to organelles for fluorescence microscopy.

• For example, if we add the signal sequence for the ER to the GFP protein then the GFP protein will be transported into the ER. Or if we add the signal sequence for the mitochondria to the RFP protein then it will be transported into the mitochondria.

• Once these fluorescent proteins are transported into the organelles, they are able to illuminate the organelle.

• This is another means by which one can make the interior of a specific organelle fluoresce.

Nuclear Pore Complex

The nuclear envelope contains an inner and outer membrane and the membrane is contiguous with the ER.

There are nuclear pores that allow for transport into the nucleus and there is also the nuclear lamina which makes the structure or interior structure of the nucleus and is made of a dense meshwork of proteins.

Schematic of nuclear pore complexes

• The nuclear pore contains cytosolic fibrils that extend along with nuclear pore complex proteins that line the rim of the nuclear pore

• The nuclear pore also contains unstructured protein loops that create a diffusion barrier. Think of this like Velcro that lines the rim of the nuclear pore preventing many molecules besides those that are relatively small from entering the nucleus.

• A nuclear pore basket is present in the interior side of the nuclear membrane which serves a similar purpose to the cytosolic fibrils that extend from the nuclear pore.

What can and cannot Diffuse In and Out of the Nucleus?

• Very small proteins (about 5kD) or molecules can passively diffuse through nuclear pores but due to the nuclear pore nature, many cannot.

• Many bigger proteins do enter the nucleus, however, through nuclear pores, but they require an active process of shuttling.

What types of molecules have to get out of the nucleus?

mRNAs, tRNAs, ribosome subunits

What types of molecules have to get into the nucleus?

Many proteins that interact with the genome, like DNA polymerase, RNA polymerase, and transcription factors.

Nuclear Import Receptors and Protein Transport to the Nucleus

Nuclear import receptors move molecules that cannot normally passively diffuse through the nuclear pores through the pores.

Prospective nuclear protein (cargo) contains a nuclear localization signal and the nuclear import receptor detects this nuclear localization signal and binds to the prospective nuclear protein to form a complex. Then this complex is able to travel into the nucleus through a nuclear pore.

How does the nuclear import receptor let go to cargo after it is bound?

However there is the question of how the nuclear import receptor lets go of the cargo protein once bound.

• Small GTP binding proteins and GTP hydrolysis drive nuclear transport.

• Ran GTPase competes with cargo for binding to the receptor.

• Once the nuclear import receptor attached to its cargo (prospective nuclear protein) enters into the nucleus, a activated Ran GTPase binds to the receptor and the protein delivered to the nucleus can disconnect from the nuclear import receptor as ran GTPase takes its place, securely locking with the nuclear import receptor.

• Once the Ran GTPase and the nuclear import receptor exits the nucleus, GTP is then hydrolyzed within the active Ran GTPase and now turned Ran GDPase dissociates from the receptor allowing the nuclear import receptor to pick up more cargo destined for the nucleus.

Nuclear Localization Signal

A nuclear localization signal (NLS) is a short sequence of amino acids that directs a protein to the nucleus. It is recognized by import proteins that help transport the protein through the nuclear pore complex.

Ran GTPase

Ran GTPase is a small GTP-binding protein that controls the direction of protein and RNA transport into and out of the nucleus. Its GTP/GDP cycle creates a concentration gradient that helps regulate nuclear import and export. Its inactive form is Ran GDPase.

It can be rendered inactive or active by Ran GEF or Ran GAP. Ran GEF switches out GDP for GTP on the Ran GTPase and then Ran GAP hydrolyzes GTP into GDP + Pi turning Ran GTPase into Ran GDPase.

Ran GEF (Guanine nucleotide exchange factor)

Ran GEF exchanges GDP for GTP turning the Ran GDPase protein into the RanGTPase protein, switching the GTPase on allowing it to being to the nuclear import receptor. This occurs in the nucleus.

Ran GAP (GTPase activating protein)

Ran GAP, associated with the cytosolic fibrils of the nuclear pore, hydrolyzes the GTP into GDP and Pi with the Pi being released. Thus Ran GTPase turns into Ran GDPase which inactivates the GTPase preventing it from continuing to bind to the nuclear import receptor. Ran GDPase, inactive, then travels back into the nucleus to be activated again and continue this cycle. This hydrolyzation occurs in the cytosol.

Transport into the Mitochondria and Chloroplasts

• Similar to the nucleus, the protein that is needed within the mitochondria is synthesized within the cytosol and needs to be delivered into the mitochondria.

• The mitochondria does not contain a nuclear pore because the intermembrane space contains a high concentration of protons and a proton gradient. A pore present would mess up this membrane potential on either side of the mitochondrial membranes.

• The transport of proteins into chloroplasts occurs through a very similar process as how it occurs in the mitochondria.

Porcess of Targeting Proteins to the Mitochondria (and by extension Chloroplasts)

Process of targeting a specific protein to the mitochondria, delivering it to the mitochondrial matrix

• Proteins synthesized for mitochondria destination contain a signal sequence that is recognized and bound to by the import receptor protein.

• Then the protein with the import receptor protein bound travels and inserts itself right near the protein translocator in the outer membrane (TOM).

• The protein is translocated (disconnects from the import receptor protein) and unfolded as it is fed through the protein translocator in the outer membrane (TOM). This allows the protein to be passed through the outer membrane of the mitochondria, but for the inner membrane the protein translocator in the inner membrane (TIM) must also feed the protein through.

• The protein is translocated across both membranes simultaneously with both the TOM and TIM complexes threading the protein into the mitochondrial matrix. This ensures that the protein is correctly transported.

• Finally once the protein is within the mitochondrial matrix, the mitochondria signal sequence within the protein is cleaved by a protease and thus the protein turns into its fully mature mitochondrial protein with folding, able to perform its function. Protein is refolded within the mitochondrial matrix.

TOM (Translocase of the Outer Membrane)

TOM (Translocase of the Outer Membrane) is a protein complex in the outer mitochondrial membrane that recognizes and transports proteins from the cytosol into the intermembrane space. It works with TIM to help deliver proteins into the mitochondrial matrix or inner membrane.

TIM (Translocase of the Inner Membrane)

TIM (Translocase of the Inner Membrane) is a protein complex in the inner mitochondrial membrane that helps transport proteins from the intermembrane space into the mitochondrial matrix or inner membrane. It works with the TOM complex to complete mitochondrial protein import.

Import Receptor Protein (Mitochondrial Protein Transfer)

An import receptor protein recognizes targeting signals on proteins destined for the mitochondria and helps guide them to the mitochondrial membrane. It is part of the machinery that transports proteins made in the cytosol into the mitochondria.

The import receptor protein bound travels and inserts itself right near the protein translocator in the outer membrane (TOM) positioning the protein its attached to correctly.

Transport into the Endoplasmic Reticulum

• Polyribosomes are bound to the ER membrane by multiple nascent polypeptide chains. This allows multiple polypeptides to be made by multiple ribosomes from the same mRNA being threaded through all of the ribosomes, allowing all of these newly formed polypeptides to be inserted into the ER lumen.

• An mRNA when transcribed forms a protein with an ER signal sequence embedded within it near the N terminus.

• Translation and translocation are coupled in a process called "co translational translocation."

• Translation by the ribosome provides the driving force needed to move the protein across the ER membrane (in this case) as it physically forces the protein to thread itself through the translocation channel

• ER protein synthesis occurs on the rough endoplasmic reticulum (RER), so-called because the endoplasmic reticulum membrane is studded with ribosomes engaged in protein translation/translocation. These proteins are translocated into the lumen of the ER and the signal peptide removed by signal peptidase.

ER Function in Protein Production and Transport

• The ER is the site of synthesis for proteins of the ER, Golgi, endosomes, lysosomes, and plasma membrane.

• The endoplasmic reticulum (ER) is the site of synthesis for proteins destined for many organelles as well as proteins to be secreted.

• The ER after it modifies the proteins through internal modification, synthesis (on the membrane and into the lumen), and folding can export those proteins via vesicles to the Golgi and then from the Golgi those proteins can go to the plasma membrane and/or be secreted or can go to the endosomes and lysosomes and other organelles

How a Protein is targeted to the Rough ER

SRP (signal recognition particle) directs a ribosome translating a protein with an ER signal sequence with a mRNA attached and a partly developed polypeptide.

• First the SRP (signal recognition particle) binds the ER signal sequence and slows/pauses translation.

• Second, the SRP bind to the SRP receptor on the ER which is right next to a translocation channel (translocon). This drags the whole SRP, undeveloped polypeptide, ribosome, and mRNA along with it.

• Third the SRP leaves or is displaced to be recycled and the ribosome engages the translocation channel with the N terminal portion of the polypeptide positioned through the translocation channel.

• Fourth, translation resumes translocating the protein across the ER bilayer and into the ER lumen. As the ribosome continues translating, the growing polypeptide is fed through the channel.

N Terminal Signal Sequence Removal

• The N terminal signal sequences are cleaved (removed) on the luminal side of the ER membrane. Basically as the polypeptide is being threaded through the translocation channel, the ER signal sequence on the polypeptide is cleaved off by the signal peptidase in the ER lumen and then this allows the protein to fold properly once it has been translocated into the ER lumen.

• Signal peptidase within the ER membrane cleaves the N terminus ER singal sequence releasing the ER protein into the lumen.

SRP (Signal recognition particle)

SRP (Signal Recognition Particle) is a complex that recognizes an N-terminal signal sequence on a newly synthesized protein and temporarily pauses translation. It directs the ribosome to the rough ER membrane, where the protein can be inserted into or transported across the ER.

SRP Receptor

SRP receptor is a protein on the ER membrane that binds the SRP–ribosome complex and brings it to the ER. It helps transfer the ribosome to a translocon, allowing the newly synthesized protein to enter or insert into the ER.

Translocation Channel (Translocon)

Translocation channel (translocon) is a protein channel in the ER membrane that allows newly synthesized proteins to enter the ER or become embedded in the ER membrane. It receives the ribosome from the SRP/SRP receptor and provides a pathway for the growing protein.

Purpose of Polypeptide Glycosylation in the ER Lumen

• Sugar chain is initially a lipid linked oligosaccharide attached to a dolichol within the ER membrane and two phosphate groups.

• Many proteins are glycosylated, or sugars are added, on certain asparagine side chains in the ER. This is called N-linked glycosylation. The protein must have a sequence where the complicated sugar tree can be attached.

• Some major functions of protein glycosylation

  • Helps protein folding and solubility

  • Protects protein in harsh environments 

  • The sugars can participate in the biological function of the protein

Signal Peptidase

Signal peptidase is an enzyme in the ER membrane that cleaves off the N-terminal signal peptide from a newly synthesized protein after it enters the ER. This allows the mature protein to continue through the secretory pathway.

Transmembrane Proteins and the ER

• Transmembrane proteins are integrated into the ER membrane during translocation.

• If there is more than one signal sequence in a protein, the first is called a start transfer sequence and the second signal is called a stop transfer sequence.

• Internal transfer signal sequences (start or stop) are not cleaved by signal peptidase. Only the initial start transfer signal sequence is cleaved when it is close to the N terminus.

• Multipass membrane proteins contain multiple stop transfer and start transfer sequences. These start and stop sequences in the polypeptide relate to translocation not translation.

• A combination of multiple stop transfer and start transfer sequences within a polypeptide will result in multipass membrane proteins. The first start transfer sequence will be cleaved off however the other start and stop sequences will remain embedded within the ER membrane.

• The hydrophobic part of the polypeptide can leave the protein translocator sideways.

Variations of ER Transmembrane Proteins and How Transmembrane Proteins In the ER are Formed

The polypeptide is threaded through the translocation channel until it reaches the stop transfer sequence at which point this threading stops. Then the signal peptidase cleaves the start transfer sequence resulting in the final polypeptide to be embedded within the ER membrane as a transmembrane protein.

• This single start transfer sequence and stop transfer sequence defines the orientation of the protein in the membrane with the N terminus in the lumen and the C terminus on the cytosolic side.

• If the placement of the polypeptide start transfer sequence is further away from the N terminus, the N terminus could be sticking out on the cytosolic side during the protein transfer. Thus, when the protein translocator reaches the stop transfer sequence, both the N and C terminus ends of the polypeptide are on the cytosolic side.

• The placement of the internal start transfer sequence can define a different orientation of the polypeptide in the membrane. The N terminus is thus not always inside the ER lumen.

If the Protein Targeting Zone is Further Away from the N terminus and there is 1 Stop Translocation Sequence what is the orientation of the final Transmembrane Protein?

Both the C and N terminus of the transmembrane protein are in the cytosol.