CELL BIO: PROTEIN SORTING

Why is protein sorting necessary?

Proteins are made mainly in the cytosol but must reach specific cellular destinations.

Approximately how many genes do humans have?

About 23,000.

About how many proteins are associated with membranes?

8,000–10,000.

Where does most protein synthesis begin?

In the cytosol.

Which organelles can synthesize some of their own proteins?

Mitochondria and chloroplasts.

Why can’t proteins simply diffuse across lipid bilayers?

The lipid bilayer is impermeable to proteins.

Why do pancreatic exocrine cells have abundant rough ER?

They specialize in secreting proteins.

What are the three protein sorting mechanisms?

Gated, transmembrane, and vesicular transport.

What is gated transport?

Movement between cytosol and nucleus through nuclear pore complexes.

Does gated transport cross a lipid bilayer directly?

No; cargo moves through nuclear pores.

What is transmembrane transport?

Movement of proteins across an organelle membrane.

Name four transmembrane transport destinations.

ER, mitochondria, chloroplasts, and peroxisomes.

What is vesicular transport?

Movement of cargo between compartments in membrane-enclosed vesicles.

Where does vesicular transport begin?

At the ER.

What is a protein sorting signal?

An amino acid sequence or patch directing a protein to a destination.

What is co-translational transport?

Protein transport occurring while the protein is being synthesized.

What are the basic steps of vesicle transport?

Budding from donor compartment, cargo and protein selection into the vesicle, vesicle movement, fusion to target compartment, and cargo delivery.

What does topologically related mean?

Proteins can move between compartments without crossing a lipid bilayer.

Which spaces are topologically equivalent?

ER lumen, Golgi lumen, vesicle lumen, and extracellular space.

What is an organelle lumen?

The interior space enclosed by an organelle membrane.

What is a luminal protein domain?

A membrane protein region facing the organelle lumen.

What is a signal sequence?

A continuous amino acid stretch recognized for sorting.

What is a signal patch?

A sorting signal formed by distant amino acids brought together by folding.

How does an ER targeting sequence typically appear?

At least eight consecutive hydrophobic, nonpolar amino acids.

Where can an ER targeting sequence occur?

At the N-terminus or internally.

What can an internal hydrophobic ER signal become?

A transmembrane domain.

What is a transmembrane domain?

A protein segment spanning a lipid bilayer.

Where is a mitochondrial targeting sequence usually found?

Near the N-terminus.

What shape does a mitochondrial targeting signal form?

An amphipathic alpha helix.

Where are chloroplast targeting signals commonly found?

Near the N-terminus.

What is the default mitochondrial import destination?

The matrix.

What is the default chloroplast import destination?

The stroma.

How do proteins reach other mitochondrial compartments?

Additional internal sorting signals direct them.

What does KDEL stand for?

Lys–Asp–Glu–Leu-COO-.

What is the function of KDEL?

Retention/retrieval of proteins belonging in the ER lumen.

Where is KDEL located?

At the C-terminus.

Why does an ER luminal protein need two signals?

An ER entry signal for import and KDEL for retention.

What does SKL stand for?

Ser–Lys–Leu.

What does SKL target?

Proteins to peroxisomes.

Where is SKL commonly located?

Near the C-terminus.

What does NLS stand for?

Nuclear localization signal.

What does an NLS do?

Directs protein import into the nucleus.

What must a NLS sequence contain?

2 Pro, 5 Basic AA. and 1 NPAA

Give an example of an NLS sequence.

Pro–Pro–Lys–Lys–Lys–Arg–Lys–Val.

Can an NLS occur within a folded protein?

Yes; it can be displayed at various positions.

What does conditional NLS exposure mean?

The signal is accessible or functional only under certain conditions.

What modification may regulate NLS exposure?

Phosphorylation.

Is the NLS usually removed after import?

No; it can remain part of the folded protein.

What surrounds the nucleus?

An inner and outer nuclear membrane.

What does NPC stand for?

Nuclear pore complex.

Where are NPCs located?

Embedded in the nuclear envelope.

What do NPCs do?

Regulate movement of molecules between nucleus and cytosol.

About how many different proteins compose an NPC?

About 30

How do small molecules (<5 kD) cross NPCs?

They can diffuse freely in both directions.

What is required for proteins larger than 60 kDa to cross NPCs?

A nuclear transport receptor escort.

What does a nuclear transport receptor recognize?

A sorting signal on cargo proteins.

How does an NLS differ from an import receptor?

NLS labels cargo; receptor escorts it through the NPC.

What are small GTPases?

Molecular switches cycling between GTP- and GDP-bound states.

Which small GTPase state is active?

GTP-bound.

Which small GTPase state is inactive?

GDP-bound.

What does GEF stand for?

Guanine nucleotide exchange factor.

How does GEF activate a small GTPase?

Promotes GDP release and GTP binding.

What does GAP stand for?

GTPase-activating protein.

How does GAP inactivate a small GTPase?

Stimulates GTP hydrolysis to GDP.

What is Ran?

A small GTPase regulating nuclear transport.

Where is Ran-GTP most abundant?

Inside the nucleus.

Where is Ran-GDP most abundant?

In the cytosol.

What does a nuclear import receptor bind?

Cargo carrying a nuclear localization signal.

Nuclear import Mechanisms.

NLS cargo binds receptor; complex crosses NPC; Ran-GTP binds receptor and releases cargo, receptor returns ro cytosol for another transport cycle.

What effect does Ran-GTP have on nuclear import cargo?

Causes the import receptor to release its cargo.

What is a nuclear export receptor?

A receptor that moves cargo from nucleus to cytosol.

Nuclear export mechanism.

Ran-GTP stabilizes receptors cargo binding site in nucleus; cargo binds to the receptor; complex exits through NPC; GTP hydrolysis releases cargo; receptor can return to nucleus.

What effect does Ran-GTP have on nuclear export cargo?

Stabilizes cargo binding to the export receptor.

Where is Ran-GEF located?

In the nucleus.

Where is Ran-GAP located?

In the cytosol.

What establishes nuclear transport directionality?

The Ran-GTP/Ran-GDP gradient maintained by Ran-GEF and Ran-GAP.

How does nuclear import differ from export?

Import: cytosol to nucleus; export: nucleus to cytosol.

Name the main mitochondrial compartments.

Outer membrane, intermembrane space, inner membrane, and matrix.

What are cristae?

Folds of the inner mitochondrial membrane.

Name the main chloroplast compartments.

Outer/inner membranes, intermembrane space, stroma, thylakoid membrane/space.

What is a mitochondrial protein translocator?

A protein complx that moves proteins across mitochondrial membranes.

What does TOM stand for?

Translocase of the outer membrane.

What does TOM do?

Recognizes targeting signals and transports proteins across the outer membrane.

What does SAM do?

Assists assembly of outer mitochondrial membrane proteins.

What does TIM23 do?

Moves proteins across the inner membrane toward the matrix.

What does TIM22 do?

Inserts certain proteins into the inner mitochondrial membrane.

What does OXA do?

Inserts proteins into the inner membrane from the matrix side.

In what folding state do proteins enter mitochondria?

Unfolded.

What does cytosolic Hsp70 do during mitochondrial import?

Prevents premature folding of precursor proteins.

What does Hsp70 stand for?

Heat shock protein 70.

What energy sources support mitochondrial protein import?

ATP hydrolysis and the H+ electrochemical gradient/membrane potential.

What drives a targeting sequence through TIM23?

The inner mitochondrial membrane potential.

What does mitochondrial Hsp70 do?

Binds incoming polypeptide and assists import into the matrix.

What is the sequence of mitochondrial matrix import?

Mitochondira precursor protein sytnhesized in cytosol → Cytosolic Hsp70 chaperones → TOM → TIM23 → mitochondrial Hsp70/ATP-driven imports target sequence.

Which proteins enter the ER?

Proteins destined for the secretory pathway.

What structures make up the ER network?

ER tubules, ER sheets, ER lumen and continuity with the outer nuclear membrane.

What is rough ER?

ER with ribosomes attached to its cytosolic surface.

What is the rough ER’s main role?

Synthesis of proteins entering the secretory pathway.

Which cell type has abundant rough ER?

Pancreatic cells

What is smooth ER?

ER without attached ribosomes.