5 - intracellular compartments and protein transport

true/false: nuclear membranes and the ER may have evolved through invagination of the plasma membrane

true

true/false: mitochondria are thought to have originated when an aerobic bacterium was engulfed by a larger anaerobic cell

true

explains why they have two membranes, have their own genomes, and don’t participate in the vesicular traffic that connects the compartments of the endomembrane system

mechanisms by which membrane enclosed organelles import proteins

transport through nuclear pores - stays folded

transport across membranes - protein unfolds

transport by vesicles - stays folded

N-terminal signal sequence

proteins destined for the ER

no signal sequence

stay in cytosol

if a signal sequence is removed from an ER protein and is attached to a cytosolic one…

both proteins are reassigned to the expected and inappropriate location

nuclear pore complex

forms a gate through which selected macromolecules and larger complexes enter or exit the nucleus

protein fibrils

protrude from both sides of the pore complex and converge to form a basket like structure on the nuclear side with spaces wide enough to not obstruct anything

prospective nuclear proteins contain a nuclear localization signal…

that is recognized by nuclear import receptors to import them from the cytosol through the nuclear pores

what drives nuclear transport?

energy from gtp hydrolysis

Ran

monomeric GTPase with two conformations

• one carrying GTP

• one with GDP

Ran-GAP

in the cytosol, triggers GTP hydrolysis and converts Ran-GTP to Ran-GDP

Ran-GEF

in the nucleus, causes Ran-GDP to release its GDP and take up GTP

localization of Ran-GAP and Ran-GEF guarantees…

the concentration of Ran-GTP is higher in the nucleus, driving the nuclear import cycle

nuclear transport process

1. nuclear import receptor picks up a prospective protein in the cytosol and enters the nucleus

2. Ran-GTP binds to the receptor, causing it to release the nuclear protein

3. the receptor (still with Ran-GTP) is transported back through the pore to the cytosol where Ran hydrolyzes its bound GTP

4. Ran-GDP falls off the import receptor

5. Ran-GDP is carried into the nucleus via its own unique import receptor

mitochondrial precursor proteins are unfolded during import bc…

a mitochondrian has an outer and inner membrane

import of mitochondrial precursor proteins

1. the mitochondrial signal sequence on a mitochondrial precursor protein is recognized by a receptor in the outer mitochondrial membrane

2. receptor is associated with a protein translocator which transports the signal sequence across the outer mito membrane to the intermembrane space

3. the complex of receptor, precursor protein, and translocator then diffuses laterally in the outer membrane until the signal sequence is recognized by a second translocator in the inner membrane

4. the two translocators transport the protein across both the outer and inner membranes, unfolding the protein in the process

5. the signal sequence is cleaved off by a signal peptidase in the mito matrix

ribosomes that are translating proteins with no ER signal sequence…

stay free in the cytosol

ribosomes that are translating proteins w/ an ER signal sequence

will be directed to the ER membrane

polyribosome

ribosomes bind to each mRNA molecule

what two factors direct a ribosome to the ER membrane?

an ER signal sequence and an SRP direct

SRP

binds to both the exposed ER signal sequence and the ribosome, slowing protein synthesis by the ribosome

SRP-ribosome complex

binds to an SRP receptor in the ER membrane. SRP is released and the ribosome passes from the SRP receptor to a protein translocator in the ER membrane. Protein synthesis resumes and the translocator starts to transfer the growing polypeptide across the lipid bilayer

a soluble protein crosses the ER membrane and enters the lumen

• protein translocator binds the signal sequence and threads the rest of the polypeptide across the lipid bilayer as a loop

• the signal peptide is cleaved from the growing protein by a signal peptidase. the cleaved sequence is ejected into the bilayer where it is degraded

• once protein synthesis is complete, the translocated polypeptide is released as a soluble protein into the ER lumen, and the protein translocator closes

a single pass transmembrane protein is retained in the lipid bilayer

• an N-terminal ER signal sequence initiates transfer

• the protein also has a second hydrophobic sequence which acts as a stop-transfer sequence

• when this sequence enters the protein translocator, the growing polypeptide chain is discharged into the lipid bilayer

• the N-terminal signal sequence is cleaved off, leaving the transmembrane protein anchored in the membrane

• protein synthesis on the cytosolic side then continues to completion

a double pass transmembrane protein has an internal ER signal sequence

• this internal sequence acts as a start-transfer signal and anchors the final protein in the membrane

• the internal signal sequence is recognized by an SRP which brings the ribosome to the ER membrane

• when a stop transfer sequence enters the protein translocator, the translocator discharges both sequences into the lipid bilayer

• neither the start transfer nor the stop transfer sequence is cleaved off, and the entire polypeptide chain remains anchored in the membrane as a double pass protein