12: Intracellular Compartments and Protein sorting

Question 1

What are the four distinct families of the intracellular compartments of eukaryotic cells?

Answer 1

1. Nucleus and cytosol
2. All organelles that function in the endocytic and secretory pathways.
   - ER, Golgi, endosomes, lysosomes, transport vesicles, and peroxisomes.
3. Mitochondria
4. Chloroplasts

Question 2

What happens to the proteins that do not contain sorting signals?

Answer 2

Remain in the cytosol as permanent residents

Question 3

Give a brief description of the three different types of protein traffic.

Answer 3

1. Gated transport - through nuclear pore complexes in the nuclear envelope.
   
   • active transport of specific macromolecules and
     free diffusion of smaller molecules
2. Protein translocation
   
   • Movement of protein s across a membrane from
     the cytosol to a topological distinct space.
   • Protein must often bu unfolded
   • Cytosol into mitochondria, plastids, ER,
     peroxisomes
3. Vesicular transport
   
   • Transport of proteins between the lumens of two
     topological equivalent compartments via vesicles.
   • E.g., ER -> Golgi

Question 4

What is a sorting signal?

Answer 4

Directs the delivery of proteins to locations outside the cytosol or to organelle surfaces.
Each sequence specifies a destination.

Stretch of aa. about 15-60 residues long.
Often found at the N-terminal of the polypeptide chain, but can be found inside the protein and used in gated transport.

Recognized by sorting receptors.
After sorting process: often removed by signal peptidases from the finished protein.

Question 5

What is the specificity of a sorting receptor?

Answer 5

Recognize classes of proteins rather than an individual protein species.
Has complementary signal sequences.

Question 6

How is the nuclear envelope composed?

Answer 6

Two continuous, concentric membranes, which are penetrated by NPCs.

Inner:
- Contains proteins that act as binding sites for chromosomes and for the nuclear lamina (protein meshwork, structural support, anchor for cytoskeleton)

Outer:

• Continuous with ER membrane.
• Has ribosomes. Proteins synthesized here are transported into the space between the two memb. (perinuclear space)

Question 7

What is a nuclear pore complex (NPC)?

Answer 7

Large multiprotein structure forming an aqueous channel (nuclear pore) through the nuclear envelope that allows selected molecules to move between nucleus and cytoplasm.

Composed of a set of about 30 different nucleoporins (proteins).

Small molecules freely diffuse through the NPC but large molecules >60 kDa require receptor-mediated mechanisms.

Question 8

What is a nuclear localization signal (NLS)?

Answer 8

Responsible for the selectivity of active nuclear import (molecules >60 kDa).
Lysine/arginine amino acid-rich stretch (+ charged) - anywhere in protein.

Must be recognized by corresponding nuclear import receptors (importins) that bind the nucleoporins of the NPC. Adaptor proteins are sometimes needed for binding.
Move along fibrils by repeatedly binding,
dissociation, and rebinding with FGrepeat sequences until inside the nucleus, where the receptor releases its cargo.

Receptors dissociate from the cargo once inside the nucleus and return to the cytosol.

Question 9

How are macromolecules and protein complexes transported out of the nucleus?

Answer 9

Nuclear export signals (NES) on macromolecules and complementary nuclear export receptors (exportins).

Receptors bind to the export signal and NPC proteins to guide their cargo through the NPC to the cytosol.
Move along fibrils by repeatedly binding,
dissociation, and rebinding with FGrepeat sequences until inside the cytosol, where the receptor releases its cargo.

Question 10

How is transport across the nuclear envelope fueled?

Answer 10

By the monomeric GTPase Ran.
Monomeric switch: active when bound to GTP, inactive when bound to GDP.

Ran-specific regulatory proteins:
Cytosolic GTPase activating protein (GAP): GTP hydrolysis, GTP -> GDP
Nuclear guanine exchange factor (GEF): GDP -> GTP

Ran-GTP (nucleus) triggers the unloading of importins and loading of exportins.

Question 11

What are shuttle proteins? Give some examples on how their transport is regulated

Answer 11

Protein that contains both nuclear localization signal (NLS) and nuclear export signal (NES). 
Fastest rate (of import/export) determines where it is mainly located. Changing the rate of import/export/both will change its localization. 

Ex. for transcription regulation:

• Phosphorylation of aa. close to signal sequence.
• Bound to inhibitory proteins that anchor them to the cytosol (e.g., cytoskeleton, organelles) or mask their NLS => no interaction with importins.

Question 12

What is the function of the nuclear lamina

Answer 12

Fibrous meshwork of proteins on the inner surface of the inner nuclear membrane. It is made up of a network of intermediate filaments formed from nuclear lamins.

Gives shape and stability to the nuclear envelope, to which it is anchored through NPCs and transmembrane proteins of the inner memb.
Interacts directly with chromatin.

Disassembles nuclear envelope in cooperation with NPCs during mitosis.

• Phosphorylated by Cdk
• When nuclear envelope memb. proteins are no longer bound to NPCs, lamina, or chromatin, they disperse throughout the ER memb. Dynein motor proteins participate.

Question 13

Which translocases (and chaperones) are important for the transport of precursor proteins into the mitochondria?

Answer 13

hsp70 - maintain precursor in unfolded state. another pulls the ppt into the organelle.

TOM - across/insertion into outer memb.
SAM - helps TOM in insertion by ensuring proper folding.
TIM23/22 - across/insertion into inner memb.
OXA - inserts proteins from matrix into inner memb.

Question 14

What are the requirements of mitochondrial translocation?

Answer 14

• Cleave off cytosolic hsp70 chaperone: ATP
• Inner membrane translocation via TIM23: H+ electrochemical gradient
• Mitochondrial hsp70 pulls polypeptide through TIM23: ATP
• Mitochondrial hsp60 bind to and release ppt in cycles of ATP hydrolysis to fold it

Question 15

Which translocases are important for the transport of precursor proteins into the chloroplast?

Answer 15

• TOC - trough outer memb.
• TIC - through inner memb.

4 routes through thylakoid memb.:

• Sec pathway
• SRP-like pathway
• TAT pathway
• Spontaneous insertion pathway (no translocases)

Question 16

Describe the transport of proteins into peroxisomes

Answer 16

Most peroxisomal proteins are made in the cytosol and inserted into the membrane of preexisting peroxisomes. Others are first integrated into the ER memb., where they are packaged into specialized peroxisomal precursor vesicles.
New precursors may fuse with each other, import/ synthesize additional peroxisomal proteins, and mature into peroxisomes.

C-terminus signal sequence signal on peroxisomal proteins is recognized by cytosolic receptors (e.g. Pex5)
=> Guide cargo to the peroxisome lumen

Multiple peroxins make up a peroxin translocator complex thought to be dynamic as they allow the import of folded and oligomeric proteins (ATP-dependent transport)

Question 17

What are the functions of the ER?

Answer 17

• Lipid and protein biosynthesis
• Intracellular Ca2+ storage, used in many cell signaling responses.
  
  • Muscle cells have modified smooth ER, the sarcoplasmic reticulum, that is specialized for Ca2+ storage. Release and reuptake trigger myofibril contraction and relaxation, respectively.

Question 18

How can the ER be isolated in the lab?

Answer 18

When tissue or cells are disrupted by homogenization, the ER breaks into fragments that reseal into small vesicles (microsomes).

Microsomes (rough/smooth) are still capable of the same functions as intracellular ER.
Ribosomes attached to rough microsomes make them more dense => equilibrium centrifugation can be used for separation.

Question 19

How can the the signal-recognition particle (SRP) bind specifically to so many different sequences? How is it structured?

Answer 19

Signal-sequence-binding site is a large hydrophobic pocket lined by methionines.
Met have unbranched, flexible side chains => pocket is sufficiently plastic to accommodate hydrophobic signal sequences of different sequences, sizes, and shapes.

Rodlike structure, wraps around the large ribosomal subunit. One end bound to ER signal seq of the partly formed ppt, other end blocks the elongation factor binding site at the interface between large/small subunits
=> halt gives ribosome enough time to bind to ER memb. before completion of the ppt, so that the ppt is not released into the cytosol. Also ensures that the protein that binds to the translocator is unfolded (no need for chaperones)1

Question 20

How does ER signal sequences and SRP direct ribosomes to the ER membrane?

Answer 20

SRP binds to the exposed ER signal seq and the ribosome => halt in transcription.

SRP receptor in ER memb. binds SRP-ribosome complex, directs it to a translocator.

SRP and SRP receptor are released. Protein is translocated.

SRP receptor has GTP-binding domains => conf. changes that occur during GTP binding/hydrolysis cycles ensure that SRP release only occurs after binding of the protein to the translocator.

Might be polyribosome binding as many ribosomes can bind to a single mRNA.

Question 21

How is the ER translocator designed?

Answer 21

Water-filled channel in the membrane.
Core: Sec61 complex with 3 subunits
- α helices of the largest subunit surround a central channel through which the ppt travels.
- Short α helix gates channel, closing translocator when it is idle => impermeable to ions.
- When open: hydrophobic aa side chains forms a seal to prevent leaking of ions.

• Can open along the seam at one sie
  => release of cleaved signal peptide into the membrane + integration of a transmemb. protein into the biayer.

Question 22

Which two modes of translocation are there for transport into the ER?

Answer 22

Co translational: most common
Movement though Sec61 occurs while protein is
being synthesized by the ribosome

Post-translational:
Protein passes through Sec61 after it is synthesized in the cytosol.
Requires a second Sec complex and BiP (a hsp70-like chaperone). ATP-dependent cycles of binding and release of BiP drive unidirectional translocation

Question 23

How is a single-pass transmembrane protein with an internal signal sequence integrated into the ER membrane?

Answer 23

Can be integrated with either the C- or N-terminus in the ER lumen depending on the distribution of the nearby charged amino acids of the start-transfer sequence.

More + before seq: C-terminus in ER lumen
More + after seq: N-terminus in ER lumen (protein must be fully synthesized)

Question 24

What is the difference between single-pass and multi-pass transmembrane proteins?

Answer 24

S: 1 start-transfer signal, 1 stop-transfer signal

M: several of both. Can be inserted in either direction. Start/stop is determined by the location of the sequence in a ppt chain. SRP starts scanning for signals from the N-terminal.
All copies of the same ppt will have the same orientation in the lipid bilayer => asymmetrical ER memb. in which the exposed protein domains on the cytosolic face differ from those at the ER lumenal .

Question 25

How do ER-resident proteins stay there without being transported to other destinations?
Give an example of one such protein

Answer 25

ER retention signal
4 aa residues on the C-terminal side

Example:
Protein disulfide isomerase (PDI): catalyzes the oxidation of free sulfhydryl (SH) groups on cysteines to form disulfide (S-S) bonds.
Disulfide bonds are common inside extracellular space/lumen of organelles.

BiP (chaperone): pulls proteins post-translationally into the ER through the Sec61 ER translocator. Hydrolyzes ATP. Recognizes incorrectly/partially folded proteins => keeps proteins from aggregating + helps keep them in the ER

Question 26

What are the functions of glycoproteins?

Answer 26

Originally simple protection, in higher organisms structural changes lead to formation of mucus, which can protect lungs from pathogen infection.

Recognition function in cell-cell adhesion (selectins).

Antigenic properties
Regulates activity of receptors (O-glycosylation of NOTCH)

Question 27

How are proteins glycosylated?

Answer 27

Formation of a precursor oligosaccharide (N-acetylglucosamine (GlcNAc), mannose, glucose) that is transferred en bloc to proteins.

Oligosaccharyl transferase has its active site exposed on the lumenal side of the ER membrane.

• Dolichol lipid anchors precursor in the ER memb.
• Sugars are activated in the cytosol by formation of nucleotide (UDP/GDP)-sugar, and transferred to dolichol. Precursor is built up on the dolichol.
• Partly through the synthesis, the lipid-linked oligosaccharide is flipped across the memb. by the help of a transporter.
• In ER: oligosaccharide “trimming”/”processing” (continues in Golgi)

Precursor is transferred to the target NH2 side chain of asparagine immediately after that aa has reached the ER lumen after protein translocation.

Question 28

Give a brief description of retrotranslocation of improperly folded glycoproteins from the ER

Answer 28

After some time, mannose is removed from the oligosaccharide by mannosidase.
Protein folds and exits ER before removal => no degradation.

Mannose removed:

• recognized by lectins and other chaperones => removal of disulfide bonds => no aggregation.
• Translocator has E3 ubiquitin ligase enz. => polyubiquitylation, marking the unfolded protein for destruction in proteasomes. Energy from ATP hydrolysis.
• N-glycanase removes oligosaccharide chains.
• Degradation

Question 29

What are the 3 different pathways of the unfolded protein response (UPR)?

Answer 29

IRE1 (transmemb. kinase):

• Regulated mRNA splicing => transcription regulation.
• Activation of genes helps mediate UPR

PERK (transmmemb. kinase):
- Phosphorylates and inhibits translation factors => protein synthesis of the cell decreases => flux into the ER decreases, reducing the number of proteins to be folded there.

ATF6 (transmembrane ER protein):

• Transcription regulator
• Increased levels of misfolded proteins => transport to Golgi, cytosolic domain cleaved off
• Transported to nucleus for activation of genes involved in UPR