Lecture 15: Intracellular Protein Transport

Question 1

Cytoplasm

Answer 1

• Cytosol + organelles

Question 2

Cytosol

Answer 2

• Makes up the bulk of the cell
• Protein synthesis/degradation
• Metabolism

Question 3

How do proteins provide the structural and functional characteristics of a given organelle?

Answer 3

• 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’

Question 4

Topological similarities

Answer 4

• Compartments whose membranes have similar compositions

Question 5

Signal sequences

Answer 5

• Protein targeting to organelles is directed by the presence of specific stretches of amino acid sequences located at the ends of proteins

Question 6

Signal patches

Answer 6

• Protein targeting to organelles is directed by the presence of specific stretches of amino acid sequences located within the protein

Question 7

What is the movement of proteins consistent with?

Answer 7

• Topological similarities among the compartments

Question 8

What is the movement of proteins mediated by?

Answer 8

• Sorting signals and receptors

- Proteins are targeted to specific organelles through the recognition of signal sequences

Question 9

Where can a signal sequence be located on the protein?

Answer 9

• Can be anywhere on the protein, including the N-terminus, C-terminus, or a 3D patch instead of a sequence

Question 10

Protein sorting receptors

Answer 10

• Signal sequences are recognized by these

- Aid in the pick up and delivery of cargo to their destination

Question 11

Gated Transport

Answer 11

• 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

Question 12

Transmembrane Transport

Answer 12

• 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

Question 13

Vesicular Transport

Answer 13

• 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

Question 14

Gated Transport #2

Answer 14

• 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

Question 15

Nuclear localization signals (NLS)

Answer 15

• 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

Question 16

Nuclear import receptors

Answer 16

• 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

Question 17

Nuclear Transport

Answer 17

• The import of nuclear proteins through the pore complex concentrates specific proteins in the nucleus.
• > Increases order in the cell
• > Consumes energy

Question 18

Ran

Answer 18

• 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

Question 19

RAN-GEF

Answer 19

• RAN guanine exchange factor (GEF)
• A nuclear protein
• Catalyses the binding of GTP to RAN inside the nucleus

Question 20

RAN-GAP

Answer 20

• RAN GTP-ase activating protein
• A cytosolic protein
• Activates hydrolysis of GTP attached to RAN

Question 21

What does the combination of RAN-GEF and RAN-GAP do?

Answer 21

• Creates a gradient of RAN-GTP across the nuclear pore – with more RAN GTP inside the nucleus than outside.
• Vice versa for RAN-GDP

Question 22

RAN-GTP

Answer 22

• 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

Question 23

RAN-GTP effect on nuclear export receptors

Answer 23

• 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

Question 24

Nuclear transport as a means of gene regulation

Answer 24

• 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

Question 25

NFAT and Nuclear Transport

Answer 25

• 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

Question 26

NFAT and Nuclear Transport #2

Answer 26

• 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

Question 27

The Transport of Proteins into Mitochondria and Chloroplasts

Answer 27

• 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

Question 28

Mitochondrial transport

Answer 28

• 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

Question 29

TOM complex

Answer 29

• 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

Question 30

TIM complexes

Answer 30

• 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

Question 31

Protein-folding chaperones

Answer 31

• 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

Question 32

Directional transport

Answer 32

• 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

Question 33

Co-translational translocation

Answer 33

• 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

Question 34

What types of proteins require co-translational translocation?

Answer 34

• 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.

Question 35

ER Signal Sequences

Answer 35

• Vary somewhat in sequence but all are: N-terminal and hydrophobic (contain 8 or more nonpolar amino acids)

Question 36

Signal Recognition Particle (SRP)

Answer 36

• 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

Question 37

SRP receptor

Answer 37

• Recognizes SRPs in the ER membrane

Question 38

Synthesis of single pass (one transmembrane region) integral membrane protein

Answer 38

• 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

Question 39

Synthesis of multi pass integral membrane protein

Answer 39

• 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