Protein Sorting

Exam 3

Where do free cytosolically translated proteins end up? When are they moved?

• cytoplasm

• nucleus

• mitochondria

• chloroplasts

• peroxisome

Never: ER, golgi, membrane, exported

Post-translational sorting: moved after the entire protein is translated

Where do membrane bound translated proteins end up? When are they moved?

• rough ER

• smooth ER

• golgi apparatus

• exported (secretion) out of cell

• transmembrane/integral membrane

• lysosome

• endosome

Never: nucleus, mitochondria

Co-translational sorting: moved to ER during translation

How do proteins indicate they aren’t supposed to stay in the cytoplasm but instead go to nucleus?

Nuclear Localization Signal!

• the protein presents a nuclear localization signal (NLS), characterized by basic amino acids (+)

• this signal is necessary (needs to be there for this to happen), and sufficient (only takes this signal for this to happen)

• this signal is not unique because multiple sets of amino acids can function as an NLS

• some NLS’s are reveals by the way a particular protein folds

This only applies to proteins translated by free cytosolic ribosomes

What gets proteins out of the nucleus?

Nuclear Export Signal!

• the protein presents an NES, characterized by being leucine rich

• this signal is both necessary and sufficient

How does the Nuclear Localization Signal (NLS) work?

1. importins recognized NLS

2. importin binds to the protein

3. once bound to importin, the protein is allowed to enter the nucleus

4. once inside the nucleus, Ran GTP bind to the other side of the importin and causes the release of the protein

5. Ran GTP is hydrolyzed, the importin releases it and is able to return to cytoplasm and resume initial function

How does the Nuclear Export Signal (NES) work?

1. exportin recognizes NES

2. exportin-Ran GTP complex binds to the protein (exportin needs Ran GTP in order to have affinity for the protein)

3. once bound, the protein is allowed to leave the nucleus

4. Ran GTP is hydrolyzed in the cytoplasm making the exportin let go of it and the protein

5. Exportin and Ran return back into the nucleus

Mitochondrial Genome

• circular

• much of the mitochondria’s DNA is now housed in the nucleus

• 16.5 kb of DNA

• 13 protein coding genes (for complex 1, 3, 4)

• 22 tRNA genes

• 2 rRNA genes

• ori

The remaining genes only code for parts of the ETC, but not everything in it! There for it is not sufficient.

Where are most mitochondrial proteins transcribed and translated?

Transcribed: nucleus

Translated: free cytosolic ribosomes

How are proteins moved into the mitochondria?

• translocon - embedded in the mitochondrial membrane allows a channel for protein entry. if not in use, it is closed. it can only open up wide enough for unfolded protein to enter

  • TIM - inner membrane transolcon

  • TOM - outer membrane translocon

• HSP 70 - chaperons in the cytoplasm that keep the protein from folding

• transit peptidase - used to remove transit sequence from protein

Steps:

1. TOM recognizes the protein’s transit sequence

2. TIM complex comes close and opens

3. head (transit sequence) moved through because the mitochondrial matrix has lower charge that the head (+)

   • electrically drawn into matix

4. chaperones are peels off during entry (required energy)

5. after entry the head is cleaves off by a protease (transit peptidase)

How are proteins moved into the rough ER?

1. the first 25 amino acids to come out of translation provide the ER signal peptide

2. ER signal is recognized by the SRP

3. SRP pauses translation (blocks A-site)

4. SRP docks with SRP receptor, and it’s only choice it to set ribosome down on translocon

5. The channel is opened and the part of the protein previously translated is let go by the SRP and pushed into the ER

6. signal peptidase (an exonuclease) cleaves target signal off

What makes up the Endomembrane System

1. rough ER

2. smooth ER

3. golgi apparatus

4. transport vesicles

5. secretory vesicles

6. lysosomes

7. endosomes