Lecture 8 - Intracellular Compartments + Protein Sorting

Question 1

Major compartments/organelles of endomembrane system of eukaryotic cells

Answer 1

• Lysosome
• Golgi apparatus
• Mitochondrion
• Endoplasmic Reticulum
• Nucleus
• Plasma membrane
• Peroxisome

Question 2

Lysosome function

Answer 2

Protein destruction

Question 3

Golgi Apparatus function

Answer 3

Protein modification and export

trans golgi faces plasma membrane

Question 4

Mitochondrion function

Answer 4

Energy production

Question 5

Endoplasmic Reticulum function

Answer 5

Smooth: lipid production, detoxification

Rough: protein production, particularly for export out of cell

Question 6

Nucleus function

Answer 6

DNA storage

Question 7

Peroxisome function

Answer 7

Lipid destruction

Question 8

Endosymbiosis theory and what organelles are present based on this.

Answer 8

Anaerobic eukaryote took in aerobic prokaryote, evolution caused aerobic bacterium to multiply w/in => anaerobic eukaryote and aerobic prokaryote form symbiotic relationship => membrane of mitochondria/chloroplast derived from eukaryotic cell host

Question 9

3 major modes of protein transport

Answer 9

1. Gated transport
2. Transmembrane transport
3. Vesicular transport

Question 10

What is gated transport?

Answer 10

The movement of proteins b/t cytosol and nucleus (nuclear pore complexes: display specificity), also import of transcription factors, nuclear factors, DNA pol, etc.

Question 11

What is transmembrane transport?

Answer 11

• Proteins transported from cytosol to mitochondria, ER, plastids, or peroxisomes
• Facilitated by TM protein translocators
• Cargo unfolds to pass through translocator

Question 12

What is vesicular transport?

Answer 12

• Proteins transported from ER to Golgi, Golgi to late endosome/lysosome/early endosome/cell exterior, cell exterior, secretory vesicles/cell exterior
• Membrane-enclosed transport intermediates ferry proteins from one compartment to another via membrane fusion.
• Proteins transported this way never cross membranes

Question 13

Properties of lipid vesicles and vesicular transport

Answer 13

Vesicles bud and fuse during vesicular transport b/c membranes share structural and physical properties

Question 14

Signal sequences: function. Don’t memorize actual AA sequences of these, though! Where located?

Answer 14

Serve as indicator of where proteins should be targeted

On N terminus

Question 15

Nuclear import from cytosol: features of the nucleus and nuclear envelope

Answer 15

Features of nucleus/nuclear envelope:
- double membrane
- contiguous w/ ER
- has nuclear pore complexes -> gateways through which things get into/out of nucleus (gated diffusion barrier via size, active transport for larger molecules)
- Nuclear lamina network of intermediate filament provide structural integrity to nucleus

Question 16

What are nuclear localization signals?

Answer 16

Signals that act as ID for molecules to enter nucleus

Question 17

What do nuclear import receptors bind?

Answer 17

NPC proteins and nuclear localization signals on cargo proteins

Question 18

Nuclear import from cytosol: role of Ran proteins, GTP hydrolysis, regulation

Answer 18

GTP-binding proteins that facilitate nuclear transport

GTP hydrolysis by Ran proteins provide directionality to nuclear transport

Regulated by type of bound guanine nucleotide => cycle b/t GDP/GTP bound forms via exchange reactions

Question 19

Nuclear import from cytosol: control of nuclear envelope formation/breakdown (slide 19).

Answer 19

• Dynamic assembly/disassembly of nuclear lamina is important for things like cell division

Question 20

Transport of proteins into mitochondrion: features, requirements, steps.

Answer 20

Two mitochondrial protein translocators:
- TOM complex: receives protein
- TIM23 complex: spans both membranes, brings protein into matrix

Requires:
- ATP hydrolysis
- Membrane potential

Steps:
- Protein has a signal sequence that binds to import receptors
- TOM Complex has receptor, triggers insertion into membrane
- TIM23 Complex translocates protein into matrix
- Signal is cleaved by signal peptidase

Question 21

Transport of proteins from the cytosol into ER: features, components, steps.

Answer 21

2 ways: Co-translational (destined for ER) or Post-translational translocation (destined elsewhere)

Co-translational:
- Ribosome translates mRNA, produced protein has signal
- SRP binds to signal peptide => pause translation
- SRP-bound ribosome attaches to SRP receptor in ER membrane
- Translation continues and translocation begins
- SRP and SRP receptor displaced/recycled

Question 22

Differences b/w SER & RER, how they can be isolated.

Answer 22

Differences: SER microsomes have low density and stop sedimenting/float at low sucrose concentration; RER microsomes have high density and stop sedimenting/float at high sucrose concentration

Separation: High speed centrifugation w/ density gradient

Question 23

The signal hypothesis of protein translocation, the SRP

Answer 23

The signal hypothesis of protein translocation: translocation is signal sequence specific, and the signal determines target compartment

Signal-recognition particle (SRP): directs ER signal sequences to a specific receptor in the RER membrane

Question 24

Translocation of soluble proteins from cyt to ER

Answer 24

• Protein w/ signal attaches to inactive protein translocator => active translocator
• Signal peptidase cleaves signal
• Mature soluble protein in ER lumen

Question 25

Translocation of single TM proteins w/ terminal ER sequence from cyt to ER

Answer 25

• Signal acts as start transfer sequence, stop-transfer sequence is internal
• Translocator translocates protein until it hits stop-transfer sequence => signal peptidase cleaves signal
• ER communicates w/ other organelles => bud off protein in membrane as vesicle to transport for destined location

Question 26

Translocation of single TM proteins w/ internal ER sequence from cyt to ER

Answer 26

• The sequence acts as both the start and stop sequence, w/ the protein orientation dependent on the location of flanking positively charged amino acids w/ respect to internal ER sequence

Question 27

Translocation of 2 TM domain proteins from cyt to ER

Answer 27

• Signal sequence is internal and acts as start-transfer, stop-transfer also internal

Question 28

Translocation of multiple TM domain proteins from cyt to ER

Answer 28

• Multiple internal start and stop sequences in protein

Question 29

Glycosylation of proteins: what is it? Functional importance?

Answer 29

• Protein modification during folding => attachment of sugars to specific AA residues

Glycoslyation can be either N-linked (asparagine) or O-linked (hydroxylate AA like serine, threonine)

Function: important for ensuring eventual folding of protein in ER lumen, if not properly folded => ubiquitination (degradation of protein)

Question 30

Degradation of misfolded proteins.

Answer 30

• Misfolded protein is brought to protein translocator
• In cytosol, ubiquitin attaches to lysine residues and protein is brought to proteasome where proteins die

Question 31

GPI-linked proteins

Answer 31

Some proteins are linked to glycosylphosphatidl-insoitol (GPI), anchoring them to membrane