Intracellular Compartments & Transport
Nuclear Localization Signals and Import
Proteins imported from the cytosol to the nucleus contain short stretches of amino acid sequences called nuclear localization signals or NLS.
Importins, specialized nuclear import receptors, recognize the NLS of cargo proteins and transport them to the nucleus through the nuclear pore complexes or NPCs.
Importins are present in the cytosol as soluble dimers of an alpha and a beta subunit. Importin alpha binds the NLS of a cargo protein to form a cargo-receptor complex.
Importin beta binds cytoplasmic fibrils extending from the NPC and docks the cargo-receptor complex onto the channel's opening.
The inner channel of the NPC is lined with phenylalanine-glycine or FG-rich repeats that form a gel-like selective barrier.
As the cargo-receptor complex moves through the channel, the beta subunit makes multiple weak contacts with the FG repeats and hops across the channel, breaking the interactions between the FG repeats and dissolving the gel-like barrier. The cargo-receptor complex travels inside the nucleus through repeated contact and dissolution.
Within the nucleus, a GTP-bound protein called Ran binds importin beta and induces a conformational change in the receptor to release the cargo protein.
The importin-Ran-GTP complex is then transported back to the cytosol, where GTP is hydrolyzed, releasing the importins for another round of cargo import.
Directing Proteins to the Rough Endoplasmic Reticulum
All proteins destined for the ER have a unique hydrophobic signal sequence at their N-terminal.
As soon as this signal sequence emerges from the ribosome, it is bound by the signal recognition particle or SRP — a ribonucleoprotein complex with a ladle-shaped structure.
In addition to a signal sequence binding pocket, SRP also has a translation pause domain and a GTP-binding domain.
The translation pause domain blocks the elongation factor binding site on the ribosome and arrests translation.
After binding to the ribosome-nascent chain or RNC complex, the SRP changes conformation, exposing a receptor binding site.
The SRP-RNC complex then uses a GTP-dependent interaction and docks at the SRP receptor present on the ER membrane.
The SRP-SRP receptor complex then carries the ribosome and the target polypeptide chain to an adjacent translocon channel.
The interaction of the SRP receptor with the translocon brings about a conformational change in the SRP,... unloading the RNC complex on the translocon.
Following unloading, GTP hydrolysis dismantles the SRP-SRP receptor complex to recycle the components for the next ER protein targeting cycle.
Insertion of Single-pass Transmembrane Proteins in the RER
The ER signal sequence of a transmembrane protein acts as a start-transfer signal for translocation through the Sec61 channel on the ER membrane.
As translocation continues, the looped signal sequence uses the lateral gate of the Sec61 channel to move out.
It’s cleaved by the adjacent signal peptidase complex, releasing the N terminal into the lumen.
A hydrophobic domain in the polypeptide chain cannot cross the lipid bilayer and acts as a stop-transfer signal.
Therefore, when the Sec61 channel encounters such a domain, it opens laterally to release the hydrophobic domain into the lipid bilayer, forming a transmembrane domain.
The ribosome then continues the synthesis of the cytosolic domain.
After translation terminates, the dissociating ribosome leaves behind a type I signal transmembrane protein embedded in the ER membrane with its N terminal in the lumen and C terminal in the cytosol.
If positively charged residues precede the hydrophobic domain, the N terminal remains out in the cytosol.
The resultant transmembrane protein, a type II protein, is an upside-down type I protein.
Introduction to Membrane Traffic
Broadly, membrane trafficking can be of three categories. Cargo can be transported within the cell from one organelle to another using the secretory pathway or, into the cell using endocytosis, and out of the cell using exocytosis.
In the secretory pathway, substances produced inside the cell are packaged into protein-coated, membrane-bound carriers called vesicles that can be transported from one organelle to another.
The SNARE family of proteins dock the vesicle to the target membrane and catalyze the fusion of the vesicle membrane to deliver the cargo.
If the vesicles fuse with the plasma membrane, the cargo is released to extracellular space, and the process is called exocytosis. Typically, substances that need to be exported are waste products, membrane proteins, or signaling molecules required for cellular communication.
Conversely, in endocytosis, substances not produced in the cell, such as vitamins, cholesterol, and micronutrients, are imported into the cell.
In pinocytosis, a type of endocytosis, a cell membrane surrounds the extracellular fluid, including water and dissolved nutrients.
Another type of endocytosis, called phagocytosis, occurs when the cell surface receptors encounter a foreign particle, usually an invading microorganism or cell debris from damaged tissue.
The cell extends to engulf the particle, and the membranes fuse, trapping the particle inside.
COP Coated Vesicles
Coated vesicles are transport vesicles that bud off from specialized regions of the cell membrane. Specific coat proteins cover their cytosolic surface.
Based on the type of protein coats, these vesicles can be of three types: coat protein or COP-coated vesicles, COPI and COPII, and clathrin-coated vesicles.
COPI vesicles transport molecules between different parts of the Golgi body and from the Golgi back to the rough ER.
COPII vesicles are formed in the ER membranes and mediate transport from the ER to the Golgi.
COPI and COPII vesicles are composed of similar coat protein complexes called coatomers. These bend the membrane to form a bud that is released from the donor membrane as a vesicle.
Clathrin Coated Vesicles
Clathrin-coated vesicles, the most well-studied coated vesicles, transport proteins from the Golgi to the plasma membrane and out of the plasma membrane for endocytosis.
The protein clathrin forms the outer layer of the coat. From the top, it appears as a three-legged triskelion structure formed from three large and three small polypeptide chains.
The triskelions assemble into a basket-like framework and determine the geometry of the clathrin cage.
The inner layer of the coat is formed by adaptor proteins that select and trap the transmembrane receptors that bind the specific molecules to be transported. The cargo and the receptor are then packaged into a newly formed clathrin-coated bud.
Dynamin, a GTP-binding protein, attaches around the neck of the bud, which triggers GTP hydrolysis. The energy derived drives a conformational change in dynamin. The neck of the bud stretches until the vesicle pinches off from the cell membrane.
SNAREs and Membrane Fusion
For a vesicle to fuse with the target organelle, the lipid layers of the two membranes must be within one point five nanometers of each other.
In this proximity, lipids flow from one bilayer to the other by displacing water molecules around the membrane. This energetically unfavorable process requires specialized fusion proteins called SNAREs.
SNAREs are transmembrane proteins with helical motifs that catalyze membrane fusion.
SNAREs exist in complementary sets in the vesicle and target membranes as v-SNARE and t-SNARE.
The helical domains of t and v-SNAREs wrap around one another to form a trans-SNARE complex that initiates membrane fusion. The energy released upon trans-SNARE complex formation locks the two membranes together.
As the cytosolic sides of the bilayers move close together, they expel the water molecules at the interface. Lipid molecules now freely flow from one leaflet to another, forming a connecting stalk.
During hemifusion, lipids in the outer leaflet mix to widen the zone of fusion. When the newly formed bilayer ruptures, fusion is complete.
SNAREs can remain in the membrane as stable complexes until they are needed for new rounds of membrane transport.
NSF, a hexameric ATPase, catalyzes SNARE disassembly. This ring-shaped protein feeds the SNARE complexes through its center to dissociate the t and v-SNAREs.