Lecture 10 Protein Sorting BIOMG 1350

How do proteins get to their correct destinations?

Proteins reach destinations via:

1) nuclear pores

2) across membranes

3) transport by vesicles (more on this in Lecture 11)

What are signal ('sorting') sequences?

Short amino acid sequences, often at the N-terminus, that direct a protein to its destination; NECESSARY and sufficient for proper targeting.

How do we know signal sequences are necessary?

Most proteins without them stay in the cytosol; adding a signal to GFP can redirect it to the ER.

What is the nuclear envelope?

Double membrane surrounding the nucleus.

How do molecules enter and exit the nucleus?

Through nuclear pores; highly structured openings in the nuclear envelope.

What EXITS the nucleus?

mRNA, ribosomal subunits, tRNA

What ENTERS the nucleus?

DNA and RNA polymerase, transcription factors

How do small proteins diffuse through nuclear pores?

Very small proteins can PASSIVELY diffuse.

How do larger proteins enter the nucleus?

Requires ACTIVE transport using a nuclear localization signal (NLS) and importin receptor.

What is importin?

A transport receptor that recognizes NLS and carries cargo through the nuclear pore complex (NPC).

How is cargo released in the nucleus?

Ran-GTP binds importin, causing release; Ran-GTP is later hydrolyzed in the cytosol to Ran-GDP, recycling importin.

What are small GTP-binding proteins (GTPases)?

Molecular switches that are ON when bound to GTP and OFF when bound to GDP.

How is a GTPase turned ON?

GDP is exchanged for GTP by a Guanine nucleotide Exchange Factor (GEF), changing its shape to active.

How is a GTPase turned OFF?

GTP hydrolysis to GDP is accelerated by GTPase-Activating Protein (GAP), inactivating the protein.

What does Ran GEF do?

In the nucleus, it converts Ran-GDP to Ran-GTP to allow importin to release cargo.

What does Ran GAP do?

In the cytosol, hydrolyzes Ran-GTP to Ran-GDP, recycling importin.

What membranes does the mitochondrion have?

Double membrane: outer and inner.

How are mitochondrial proteins imported?

Fully synthesized in the cytosol; N-terminal Mitochondrial Targeting Sequence (MTS) recognized by TOM complex on outer membrane.

What is TOM?

Translocase of the Outer Membrane; binds the MTS and translocates protein into the intermembrane space.

What is TIM?

Translocase of the Inner Membrane; TIM23 imports proteins into matrix or inner membrane; TIM22 inserts multi-pass proteins.

What provides energy for mitochondrial import?

1) ATP used by cytosolic chaperones to keep protein unfolded, 2) Membrane potential across inner membrane pulls positively charged presequence through TIM.

When is MTS removed?

By peptidase once protein reaches the matrix; then protein folds with chaperones.

Do proteins need to be unfolded to enter mitochondria?

Yes, the channel is narrow; fully folded proteins cannot pass.

Do chloroplasts use a similar import mechanism?

Yes, very similar.

What proteins are synthesized in the ER?

Proteins for ER, Golgi, endosomes, lysosomes, plasma membrane, and secretion.

What is required for ER targeting?

N-terminal ER signal sequence.

What is the SRP?

Signal Recognition Particle; binds ER signal sequence as protein is translated, pausing translation and targeting ribosome to ER.

What is the translocon?

Channel in ER membrane through which growing protein is threaded into the ER lumen.

What is co-translational translocation?

Protein is moved into ER while being synthesized by ribosome.

What happens to the signal sequence in ER?

Cleaved by signal peptidase.

What modifications occur in ER?

Folding and glycosylation (sugar addition on asparagine residues).

How are transmembrane proteins integrated?

Internal signal/stop-transfer sequences anchor protein in ER membrane; N-terminus in ER, C-terminus in cytosol; alpha-helices form transmembrane domains.

Are internal signal sequences cleaved?

No, they remain as part of the transmembrane domain.

How do multipass membrane proteins work?

Contain multiple start- and stop-transfer sequences to determine topology.

What is the function of glycosylation in the ER?

Helps folding, solubility, protects protein, may aid sorting; occurs only in ER, never cytosol.