Lecture 15: Intracellular Protein Transport Flashcards
1
Q
Cytoplasm
A
- Cytosol + organelles
2
Q
Cytosol
A
- Makes up the bulk of the cell
- Protein synthesis/degradation
- Metabolism
3
Q
How do proteins provide the structural and functional characteristics of a given organelle?
A
- Catalyzing organelle-specific reactions within the organelle lumen
- Selectively transporting small molecules into and out of the lumen
- Serving as surface markers that identify the organelle and direct new deliveries of protein ‘cargo’
4
Q
Topological similarities
A
- Compartments whose membranes have similar compositions
5
Q
Signal sequences
A
- Protein targeting to organelles is directed by the presence of specific stretches of amino acid sequences located at the ends of proteins
6
Q
Signal patches
A
- Protein targeting to organelles is directed by the presence of specific stretches of amino acid sequences located within the protein
7
Q
What is the movement of proteins consistent with?
A
- Topological similarities among the compartments
8
Q
What is the movement of proteins mediated by?
A
- Sorting signals and receptors
- Proteins are targeted to specific organelles through the recognition of signal sequences
9
Q
Where can a signal sequence be located on the protein?
A
- Can be anywhere on the protein, including the N-terminus, C-terminus, or a 3D patch instead of a sequence
10
Q
Protein sorting receptors
A
- Signal sequences are recognized by these
- Aid in the pick up and delivery of cargo to their destination
11
Q
Gated Transport
A
- One of the three fundamental mechanisms of transport between cellular compartments
- Protein traffic between the cytosol and nucleus (topologically similar compartments)
- Occurs through nuclear pore complexes
- Function as selective gates that actively transport specific macromolecules and macromolecular assemblies
- Also allow free diffusion of smaller molecules
12
Q
Transmembrane Transport
A
- One of the three fundamental mechanisms of transport between cellular compartments
- Protein traffic between the cytosol and an organelle that is topologically different
- Occurs through membrane-bound protein translocators
- The transported protein molecule usually must unfold to snake through the translocator.
- Example: Cytosol —> ER
- Example: Cytosol —> mitochondria
13
Q
Vesicular Transport
A
- One of the three fundamental mechanisms of transport between cellular compartments
- Protein traffic among topologically equivalent organelles
- Occurs through membrane-enclosed transport intermediates called VESICLES
- ER↔ Golgi
- Golgi ↔ Endosomes
- Endosomes↔ Lysosomes
- Endosomes ↔ Plasma Membrane
14
Q
Gated Transport #2
A
- Proteins move through specialized nuclear pores that selectively transport macromolecules/complexes into and out of the nucleus.
- These pores do allow the free diffusion of smaller molecules
- Nucleoporins lining the central pore contain unstructured regions that act to restrict the passage of large macromolecules
- > Something the size of a ribosome can be transported into the nucleus upon receiving a signal
15
Q
Nuclear localization signals (NLS)
A
- Are within the cargo being transported into the nucleus
- Must be recognized in order to initiate transport into the nucleus
- Specific nuclear localization signal sequences (NLSs) are present only in nuclear proteins
- > Characteristic is that 5 basic amino acids in a row
- > The sequence isn’t important, but the presence of the 5 amino acids are
- > Fluorescence microscopy can confirm this, as if one of the amino acids were to be mutated to a non-basic amino acid, the protein stops being nuclear and becomes cytoplasmic
16
Q
Nuclear import receptors
A
- Recognize nuclear localization signals (NLS) to initiate transport into the nucleus
- Encoded by a family of related genes to nuclear localization signals (NLS)
- Different import receptors are going to import different cargo proteins, but all work by the same mechanism
17
Q
Nuclear Transport
A
- The import of nuclear proteins through the pore complex concentrates specific proteins in the nucleus.
- > Increases order in the cell
- > Consumes energy
18
Q
Ran
A
- A small GTPase that is thought to provide the energy for nuclear transport by the hydrolysis of GTP
- Hydrolizes GTP in the process of moving proteins across the nuclear membrane
- Found in both the cytosol and the nucleus, and it is required for both the nuclear import and export systems
- Exists in two states: One with GTP attached and One with GDP attached
19
Q
RAN-GEF
A
- RAN guanine exchange factor (GEF)
- A nuclear protein
- Catalyses the binding of GTP to RAN inside the nucleus
20
Q
RAN-GAP
A
- RAN GTP-ase activating protein
- A cytosolic protein
- Activates hydrolysis of GTP attached to RAN
21
Q
What does the combination of RAN-GEF and RAN-GAP do?
A
- Creates a gradient of RAN-GTP across the nuclear pore – with more RAN GTP inside the nucleus than outside.
- Vice versa for RAN-GDP
22
Q
RAN-GTP
A
- Binds to nuclear import receptors after they diffuse through the nuclear pore and into the nucleus
- Causes them (nuclear import receptors) to release their cargo proteins, which therefore accumulate inside the nucleus
- The nuclear import receptors then leave the nucleus to then have RAN-GTP be hydrolyzed to RAN-GDP by RAN-GAP
- The nuclear import receptor then releases RAN-GDP, and then it will bind to another cargo protein and repeat the cycle
23
Q
RAN-GTP effect on nuclear export receptors
A
- Has the opposite effect on nuclear export receptors, causing them to bind their cargo.
- They then diffuse through the pore into the cytosol, where RAN-GAP will hydrolize RAN-GTP to RAN-GDP
- This causes the nuclear export receptors to release their cargo and RAN-GDP, and then cycle back into the nucleus to repeat the cycle
24
Q
Nuclear transport as a means of gene regulation
A
- The activity of some gene regulatory proteins is controlled by keeping them out of the nuclear compartment until they are needed there.
- In many cases, this control depends on the regulation of nuclear localization and export signals; these can be turned on or off, often by phosphorylation of adjacent amino acids
25
Q
NFAT and Nuclear Transport
A
- NFAT is involved in immune signaling
- T cell activated via antigen binding
- This leads to a Ca2+ levels increase due to Ca2+ channel opening
- This rise in Ca2+ levels activates the protein phosphatase, calcineurin
- Calcineurin then dephosphorylates NF-AT
26
Q
NFAT and Nuclear Transport #2
A
- Dephosphorylation of NFAT causes a conformational change which exposes a nuclear import sequence on the protein’s surface.
- NFAT enters the nucleus through gated transport, where it triggers gene expression appropriate to the T-cells role in the immune response.
- NFAT can be turned off by being rephosphorylated in the nucleus, which hides the nuclear import signal and reveals the nuclear export signal
- NFAT is then shuttled out of the nucleus to repeat the cycle
27
Q
The Transport of Proteins into Mitochondria and Chloroplasts
A
- Neither the mitochondrial nor chloroplast genomes contain information necessary to code for all of their proteins
- They rely on the import of their proteins from the cytosol following synthesis
28
Q
Mitochondrial transport
A
- Mitochondrial proteins are first fully synthesized as precursor proteins in the cytosol and then translocated into mitochondria.
- Most of the mitochondrial precursor proteins have a signal sequence at their N terminus that, when folded forms an amphipathic alpha helix.
- Charged residues cluster on one side
- Uncharged residues cluster on the other side
- The conformation of the alpha helix is what is recognized, not the actual amino acid sequence
29
Q
TOM complex
A
- Translocator of the Outer Membrane
- Functions across the outer membrane
- ALL nucleus-encoded mitochondrial proteins must first enter via TOM
- Helps insert transmembrane proteins into outer mitochondrial membrane
- Transmembrane proteins with a β-barrel structure are transferred to the SAM complex for proper folding
30
Q
TIM complexes
A
- Two complexes: TIM23 and TIM22
- Function across the inner membrane
- TIM 23 spans both outer and inner mitochondrial membranes
- Transports: soluble proteins into MATRIX & membrane proteins into inner mitochondrial membrane
- Import ATPase complex binds to and pulls proteins through TIM23 channel
31
Q
Protein-folding chaperones
A
- Prevent newly synthesized (precursor) Mitochondrial Proteins in the cytosol from aggregating
- Most common chaperone: Hsp70
- Mitochondrial versions of these chaperones also exist and help these precursor proteins fold into 3D structures once they enter the mitochondria
32
Q
Directional transport
A
- Not energetically favorable -requires energy
- Mitochondrial protein import requires ATP
- Cytosolic chaperone disassembly driven by ATP hydrolysis
- Pulling of protein through inner membrane is driven by an electrochemical gradient and ATP hydrolysis
33
Q
Co-translational translocation
A
- What proteins entering the ER undergo
- Imported into the ER as they are being synthesized
- Energy comes from the amino acids being added to the protein
- All proteins requiring co-translational translocation possess an ER Signal Sequence
34
Q
What types of proteins require co-translational translocation?
A
- Water soluble (non membranous) proteins destined to:
- > Localize to the lumen of any non nuclear organelle (ER, Golgi, lysosomes, etc.) (except the mitochondria and chloroplasts)
- > Be secreted out of the cell (e.g. hormones)
- > ER signal sequences are cleaved by a signal peptidase following translocation
- Transmembrane proteins destined to:
- > Localize to the membrane of an organelle: nuclear membrane (some), plasma membrane, ER, Golgi, lysosomal, etc.
35
Q
ER Signal Sequences
A
- Vary somewhat in sequence but all are: N-terminal and hydrophobic (contain 8 or more nonpolar amino acids)
36
Q
Signal Recognition Particle (SRP)
A
- Recognizes ER signal sequences
- Are complex proteins in that they contain both RNA and polypeptide components
- > RNA portion blocks elongation factor binding site (elongation factors bring in new tRNAs)
- > Polypeptide component binds signal sequence
37
Q
SRP receptor
A
- Recognizes SRPs in the ER membrane
38
Q
Synthesis of single pass (one transmembrane region) integral membrane protein
A
- The signal /start transfer sequence is cleaved
- An additional, hydrophobic stop transfer sequence anchors protein in the membrane
- More positively charged amino acids BEFORE the hydrophobic start/signal
- > The side with the positive charge will face the cytosol and the side with the negative charge will face the ER lumen
- > The N terminus will face the cytosol and the C terminus will face the ER lumen
- More positively charged amino acids AFTER the hydrophobic start/signal
- > The N terminus will face the ER lumen and the C terminus will face the cytosol
39
Q
Synthesis of multi pass integral membrane protein
A
- There are multiple start and stop transfer sequences
- Each hydrophobic patch represents a transmembrane region
- The first start sequence is the signal sequence
- If there is an odd number of transmembrane regions, then the N terminus and C terminus will be on opposite sides
- If there is an even number of transmembrane regions, then the N terminus and C terminus will be on the same side
