BB 314 Unit 5: Protein Sorting

Nuclear Lamina

A dense fibrillar network inside the nucleus of most cells. It is composed of intermediate filaments and membrane associated proteins. Besides providing mechanical support, the nuclear lamina regulates important cellular events such as DNA replication and cell division.

Know examples of molecules that are transported into and out of the nucleus, and why.

-mRNA -???

Ran-GTP and Ran-GDP

A protein called Ran, which has a GTP molecule bound to it, binds to the receptor-cargo complex. This makes the importin release the cargo protein into the nucleus. The importin, still bound to Ran-GTP returns to the cytosol, where Ran-GTP is released from the importin and the GTP bound to Ran is hydrolyzed to GDP. In this way, the importin is brought back to the cytosol where it can pick up another protein destined for the nucleus.

Signal sequence

A short peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. These proteins include those that reside either inside certain organelles (the endoplasmic reticulum, golgi or endosomes), secreted from the cell, or inserted into most cellular membranes.

Signal recognition particle /SRP receptor

An abundant, cytosolic, universally conserved ribonucleoprotein (protein-RNA complex) that recognizes and targets specific proteins to the endoplasmic reticulum in eukaryotes and the plasma membrane in prokaryotes.

Nuclear Pore complex

Channel through which molecules like RNA and protein can travel from nucleus to cytosol or the other way around.

How is cholesterol is delivered to cells by receptor mediated endocytosis?

Cholesterol is transported through the bloodstream in the form of lipoprotein particles, the most common of which is called low-density lipoprotein, or LDL. Studies in the laboratories of Michael Brown and Joseph Goldstein demonstrated that the uptake of LDL by mammalian cells requires the binding of LDL to a specific cell surface receptor that is concentrated in clathrin-coated pits and internalized by endocytosis. As discussed in the next section, the receptor is then recycled to the plasma membrane while LDL is transported to lysosomes, where cholesterol is released for use by the cell.

Clathrin

Coat proteins. Once the vesicle has budded off, the clathrin coat is removed and the vesicle can be transported to the target organelle

Constitutive and regulated secretion

Constitutive Secretion: proteins are secreted from a cell continuously, regardless of external factors or signals. This pathway is also used by all eukaryotic cells to maintain the cell's plasma membrane. Regulated Secretion: proteins are secreted from a cell in large amounts when a specific signal is detected by the cell.

What is the function of the lysosome?

Digest excess or worn out organelles, food particles, and engulfed viruses or bacteria.

How are soluble proteins delivered into the ER lumen, and how are membrane proteins inserted into the membrane of the ER (what targets a protein to the ER, what other proteins are necessary, what role do they play, what features of proteins result in their insertion into a membrane, etc.).

Endoplasmic Reticulum • For most proteins that end up in the ER, the ribosomes on which these proteins are being made are brought to the surface of the ER, and the proteins are sent into the lumen of the ER as they are being synthesized. • As the signal sequence of a protein is made by the ribosome and emerges from it, the sequence is recognized and bound by a signal recognition particle (SRP). The SRP, in turn is recognized and bound by a receptor on the surface of the ER. This SRP receptor is situated near a channel in the ER membrane, so when the SRP binds a ribosome making a protein to be delivered to the ER, the ribosome is positioned over the channel. • Once the ribosome has been brought to the surface of the ER, it binds to a protein translocation channel in the ER membrane and inserts the signal sequence into the ER lumen. The ribosome then continues translation, effectively pushing the growing polypeptide into the lumen of the ER. Once the ribosome is docked in place on the ER translocation channel, the SRP dissociates from the complex and wanders off to search for another signal sequence.

Which proteins (i.e., destined for which cellular destination) are made on free ribosomes and which are made on membrane-bound ribosomes?

Free Ribosomes: Proteins that function within the endomembrane system (such as lysosomal enzymes) or those that are destined for secretion from the cell (such as insulin) are synthesized by bound ribosomes. Membrane-Bound Ribosomes: In contrast, proteins destined to remain in the cytosol or to be incorporated into the nucleus, mitochondria, chloroplasts, or peroxisomes are synthesized on free ribosomes and released into the cytosol when their translation is complete.

What are the major cellular functions of the nucleus?

It stores the cell's hereditary material, or DNA, and it coordinates the cell's activities, which include growth, intermediary metabolism, protein synthesis, and reproduction

KDEL sequence

KDEL is a target peptide sequence in the amino acid structure of a protein which prevents the protein from being secreted from the endoplasmic reticulum (ER). A protein with a functional KDEL motif will be retrieved from the Golgi apparatus by retrograde transport to the ER lumen.[1] It also targets proteins from other locations (such as the cytoplasm) to the ER. Proteins can only leave the ER after this sequence has been cleaved off.

Dynamin

Large GTPase implicated in the budding and scission nascent vesicles from parent membranes.

What proteins are sent to the lysosome?

Lysosomal Proteins

Mannose-6-phosphate and M 6-P receptor

Mannose-6-phosphate: The mannose-6-phosphate is recognized by special cargo receptor proteins in the trans Golgi membrane that then binds the lysosomal proteins and sends them to the lysosome, packaged in vesicles. M 6-P receptor: a protein that spans the membrane, one part being inside the lumen of the Golgi, where it binds the lysosomal protein and the other end in the cytosol. The cytosolic side of the receptors is recognized and bound by proteins called adaptins. The clathrin network is formed by the binding of clathrin to the adaptins.

Explain by drawing a flowchart or figure how proteins are imported into the nucleus (what targets a protein to the nucleus, what other proteins are necessary, what role they play).

NLS and Ran-GTP involved.

Where in the cell are proteins made and how they are delivered to the cellular compartments in which they work?

Proteins are assembled from mRNA by ribosomes in the cytoplasm. Protein signals are the address labels of the proteins.

Explain the mechanism of transport of proteins from the Golgi complex to lysosomes (what signal targets proteins to the lysosomes, what other proteins are required for the transport of proteins to the lysosomes, what are their roles?)

Proteins that are to be sent to the lysosome receive special treatment in the Golgi complex. When these proteins enter the cis Golgi, they are phosphorylated on the specific mannose sugars attached to them. This results in the formation of mannose-6- phosphates on the lysosomal proteins. The enzyme that adds phosphate to the mannose can recognize proteins that are destined for the lysosome. The mannose-6-phosphate is recognized by a special receptor protein in the trans Golgi that then binds the lysosomal proteins and sends them to the lysosome.

Nuclear Transport Receptors

Proteins that exhibit a high affinity for a small GTPase, called Ran, in the GTP bound form

Nuclear Localization sequence

Serves as the label that marks the protein for delivery to the nucleus.

Signal peptidase

Signal peptidases are enzymes that convert secretory and some membrane proteins to their mature forms by cleaving their signal peptides from their N-terminals.

Rough and smooth ER

Smooth ER: makes cellular products like hormones and lipids. Rough ER: important in the synthesis and packaging of proteins.

How do proteins going to different cellular compartments get into the target organelle (e.g., through the nuclear pore complex, through translocation channels, in vesicles, etc.)?

Soluble proteins in the lumen of the ER are packed up in membrane vesicles that then travel to the next destination, where the vesicles fuse with the membrane of the target organelle, emptying the contents of the vesicle into the target. Nuclear Pore Complex: NLS and Ran-GTP involved. Translocation channels: transports nascent polypeptides with a targeting signal sequence into the interior (cisternal or lumenal) space of the endoplasmic reticulum (ER) from the cytosol. Vesicles: Those that are travelling beyond the ER move from the ER to the Golgi, and from there to the outside of the cell in secretory vesicles, if they are secreted proteins. If they are intended for the lysosome, the vesicles fuse with the lysosome to deliver them

How do transport vesicles form?

The formation of vesicles is triggered by the assembly of a lattice or network of special proteins on the cytosolic side of the membrane. There are several different kinds of these so-called coat proteins, of which the best studied is called clathrin. Clathrin molecules start vesicle formation by forming a net over a part of the membrane and pinching off a region of the membrane. The vesicle is sealed shut with the help of another protein, dynamin.

Trans and Cis Golgi network

Trans: the final cisternal structure, from which proteins are packaged into vesicles destined to lysosomes, secretory vesicles, or the cell surface. The TGN is usually positioned adjacent to the stacks of the Golgi apparatus, but can also be separate from the stacks Cis: collection of fused, flattened membrane-enclosed disks known as cisternae (singular: cisterna, also called "dictyosomes"), originating from vesicular clusters that bud off the endoplasmic reticulum The vesicles that leave the rough endoplasmic reticulum are transported to the cis face of the Golgi apparatus, where they fuse with the Golgi membrane and empty their contents into the lumen

Importin

Type of nuclear transport receptor. The importin binds to the NLS and brings the protein to be transported to the nuclear pore. The complex consisting of the importin and its "cargo" then moves through the nuclear pore into the nucleus.

Stop-transfer sequence

When a peptide containing a stop-transfer sequence inserts into the channel, the translocation process is interrupted. Subsequently, the translocation channel releases the polypeptide chain carrying the stop-transfer sequence laterally to the lipid bilayer. The stop-transfer sequence thus becomes a transmembrane segment

Receptor mediated Endocytosis

a process by which cells absorb metabolites, hormones, other proteins - and in some cases viruses - by the inward budding of plasma membrane vesicles containing proteins with receptor sites specific to the molecules being absorbed

Acid hydrolases.

an enzyme that works best at acidic pHs. It is commonly located in lysosomes, which are acidic on the inside. Catalyzes the hydrolysis of a chemical bond.

Recycling receptors

recognize the KDEL sequences and bind the proteins that have these sequences. The recycling receptors are then selectively transported back to the ER in vesicles, with the KDEL proteins bound to them.

What is the path of secreted proteins from the ribosome to the exterior of the cell?

rough ER → Golgi → secretory vesicles → cell exterior.

Nuclear Envelope

the lipid bilayer membrane which surrounds the genetic material and nucleolus in eukaryotic cells.

What is responsible for the specificity of vesicular transport? (that is, what ensures that vesicles deliver their cargo to the correct compartments?)

v-SNAREs and t-SNAREs

v-SNAREs and t-SNAREs

v-SNAREs: On the surface of a transport vesicle. t-SNAREs: On the surface of a target organelle. Each organelle and each type of transport vesicle is believed to have unique SNAREs. This accounts for the high specificity of vesicular transport. Transport vesicles in cells display on their surfaces molecular markers that must be recognized by receptor molecules on the target organelle before the vesicle can fuse with the target and deliver the contents.

Membrane-bound and free ribosomes

Membrane-Bound: On the ER Free Ribosome: In the cytosol

Adaptin

The cytosolic side of the receptors is recognized and bound by proteins called adaptins. Proteins that mediate the formation of vesicles for intracellular trafficking and secretion

Which proteins are sent to the rough ER and why (what happens to the proteins in the RER?).

Sorting of proteins destined for various other locations in the cell, like the Golgi apparatus, the lysosomes and the plasma membrane. As already discussed, the signal sequence of the protein is cleaved and the protein is released into the lumen of the ER. A protein released into the ER may stay in the ER, or it may travel, via the Golgi, to the lysosomes or plasma membrane or be secreted. But regardless of where it ends up finally, the protein may have various things happen to it in the lumen of the ER. These include: -Folding -Assembly into multisubunit complexes -Disulfide bond formation -Glycosylation (addition of some sugars) -Addition of glycolipids to some proteins that are destined for the plasma membrane.