BSCI331: Final Exam - Intracellular Protein Transport

the movement of proteins between organelles is consistent with ____ among these compartments

topological similarities

topological similarities

compartments with similar membrane orientations

signal patch (3D)

• higher order of structure

• secondary and tertiary structures of amino acids that originally are not near from each other get brought close to each other

three fundamental mechanisms of the movement of proteins between cellular compartments:

1. gated transport

2. transmembrane transport

3. vesicular transport

gated transport

• protein traffic between the cytosol and nucleus → cytosol to nucleus and/or nucleus to cytosol

• occurs through nuclear pore complexes from cytosol to nucleus without stepping off of leaflet

• function as selective gates → actively transport specific macromolecules and macromolecular assembles

• allow free diffusion of smaller molecules

transmembrane transport

• thread protein through usage of transporter for proteins to move from one face to another face

• protein traffic between the cytosol and an organelle that is topologically different

• occurs fthrough membrane-bound protein translocators

• transported protein molecule usually must unfold to snake through translocator

• ex: cytosol → ER; cytosol → mitochondria

vesicular transport

• outside of cell is topologically equivalent to the inside

• one side of leaflet or membrane of a compartment faces outside of cell and/or other may face toward cytosol

• protein traffic among topologically equivalent organelles

• occurs through membrane-enclosed transport intermediates called vesicles

• ex ER ←→ Golgi; Golgi ←→Endosomes; Endosomes ←→ Lysosomes; Endosomes ←→ Plasma Membrane

vesicles

• bud from one organelle then fused to or from another membrane

• merging lipid membranes

nucleoporins

lining the central pore contain unstructured regions that act to restrict the passage of large macromolecules, only let small molecules through from the cytosol into the nucleus

what size do the molecules have to be in order to enter the nucleus by free diffusion to enter through the nucleoporins?

up to 9 nm diameters

what size do these kind of molecules have to be in order to enter the nucleus by active transport to enter through the nucleoporins? what steps are necessary in order for them to enter through the nucleoporins?

• upon receiving a signal, channel can open to 30 nm wide

• macromolecules where a conformation change has to happen for them to pass through the nucleoporins

nuclear localization signals (NLS)

• appear within end site of cargo which are recognized by nuclear import receptors (aka carrier proteins)

• 5 or more basic amino acids in a row in nuclear import protein and/or NLS in order to fit as a NLS site

does the import of nuclear proteins through the pore complex increase order in the cell or decrease it that impacts the concentration of specific proteins in the nucleus?

increases order in the cell → consumes energy due to a decrease in entropy

Ran

• small GTPase found in both cytosol and nucleus

• required for both nuclear import and export systems

RAN, like other GTP-binding proteins, exists in two states:

• one with GTP attached (“on” state)

• one with GDP attached (“off” state)

RAN-GEF (RAN guanine exchange factor (GEF))

• nuclear protein, binds to chromatin inside of cell

• catalyzes binding of GTP to RAN inside the nucleus

• RAN-GDP found in cytosol

RAN-GAP (RAN GTP-ase activating protein)

• cytosolic protein, outside of cell

• activates hydrolysis of GTP attached to RAN to hydrolyze to GDP

• creates gradient of RAN-GTP across nuclear pore with more RAN GTP inside nucleus than the outside

• RAN-GTP found in nucleus

role of nuclear import receptor when RAN binds:

• unicellular and mutually exclusive → when RAN-GTP binds to nuclear import receptor then cargo protein won’t be able to bind

• cargo protein will bind to import receptor from the cytosol to be allowed to enter inside of nucleus → cargo lets go where RAN-GTP comes and binds to import receptor → enter out of cytosol and gets hydrolyzed into RAN-GDP which allows cargo protein to bind to it again

• binds to import receptor after they diffuse through nuclear pore and into nucleus

• causes them to release their cargo proteins, which therefore accumulate inside the nucleus

role of nuclear export receptor when RAN binds:

• not mutually exclusive → would need both RAN-GTP and cargo protein binded to export receptor to carry reaction

• nuclear export receptor needs to be naked to enter in cytosol → both cargo protein and RAN-GTP both bind on either end sites of receptor → goes back out into cytosol and gets hydrolyzed into RAN-GDP which releases RAN-GTP and cargo protein

• RAN-GTP has opposite effect on export receptors, causing them to bind their cargo

• then diffuse through pore into the cytosol

in mitochondrial transport, what are mitochondrial proteins?

• first fully synthesized as precursor proteins in cytosol and then translocated into mitochondria

• requires secondary structure, rely on chemical nature of amino acids and not specific amino acids

• AMPHIPATHIC nature → one side or face has charged residues clustered, while other side has uncharged residues clustered (represent as signal sequences)

• most of the mitochondrial precursor proteins have signal sequence at their N terminus that, when folded forms an amphipathic alpha helix

Protein translocation across mitochondrial membranes is mediated by multi-subunit protein complexes that function as protein translocators:

• TOM complex (translocase of outer mitochondrial membrane)

• two TIM complexes (translocase of inner mitochondrial membrane) → TIM23 and TIM22

TOM complex (translocase of outer mitochondrial membrane)

• ALL nucleus-encoded mitochondrial proteins must first enter via TOM → must be UNFOLDED in order to enter through

• helps insert transmembrane proteins into outer mitochondrial membrane, gatekeeper of mitochondrion

• transmembrane proteins with a B (beta)-barrel structure are transferred to the SAM (functions sorting and assembly) complex for proper folding

TIM complex (translocase of inner mitochondrial membrane)

• TIM23 spans both outer and inner mitochondrial membranes → has extension from inner to outer membranes that allow TIM and TOM to interact for proteins to enter through matrix and threaded through series

• Transports: soluble proteins into MATRIX; membrane proteins into inner mitochondrial membrane

• import Hsp70 import ATPase complex binds to and pulls proteins through TIM23 channel → chaperone protein used to clear out unstable/unfolded proteins, pulls protein across inner membrane channel by hydrolyzing ATP, increase order of cell from unfolded to fold into 3D shape

does the protein have to be folded or unfolded to go through the TOM complex?

unfolded because it needs to fit in order to thread through and then later folding up again

Hsp70 ATP-ase chaperone

• newly synthesized (precursor) mitochondrial proteins in cytosol are surrounded by protein-folding chaperones that prevent them from aggregating

• mitochondrial versions of these chaperones also exist and help these precursor proteins fold into 3D structures once they enter the mitochondria

• part of TIM complex and known to be most common chaperone

• instead of assisting in folding protein it prevents folding to happen → ensure proteins don’t fold properly to allow them to cross through mitochondrial membrane

• need to hydrolyze ATP to unfold proteins

Co-translational translocation

• imported into ER as they are being synthesized at the same time

• helps to save much effort in unfolding protein

What types of proteins require co-translational translocation?

• water soluble (non membranous) proteins destined to: localize to the lumen of any non nuclear organelle (ER, Golgi, lysosomes, etc.); be secreted out of the cell (e.g. hormones) → found in ER first, don’t need protein translocator to secrete proteins since ER lumen topologically similar to extracellular fluid

• transmembrane proteins destined to: localize to the membrane of an organelle (some nuclear membrane, plasma membrane, ER, Golgi, lysosomal, etc.); located inside membrane or plasma membrane → translocated in ER

water soluble (non membranous) proteins

• localize to the lumen of any non nuclear organelle (ER, Golgi, lysosomes, etc.)

• be secreted out of the cell (e.g. hormones) → found in ER first, don’t need protein translocator to secrete proteins since ER lumen topologically similar to extracellular fluid

transmembrane proteins

• localize to the membrane of an organelle (some nuclear membrane, plasma membrane, ER, Golgi, lysosomal, etc.)

• located inside membrane or plasma membrane → translocated in ER

ER signal sequence

• ALL proteins requiring co-translational translocation possess a ER signal sequence

• defined from chemical nature and based on 5 or more basic amino acids (similar to NLS or nuclear localization signalling)

• vary somewhat in sequence but all are: n-terminal, hydrophobic → contain 8 or more hydrophobic/nonpolar amino acids (about the width of one lipid bilayer of ER)

• signal sequences recognized by a signal recognition particle (SRP)

• recognized by an SRP receptor in the ER membrane

SRP (signal recognition particle)

• non-coding RNA

• recognizes ER signal sequences in co-translational translocation

• complex proteins in that they contain both RNA and polypeptide components

how does a SRP play a role for the SRP RNA molecule?

1. red part → RNA portion end that blocks elongation factor binding site (elongation factors bring in new tRNAs); binds import channel to preven bringing in new tRNAs into ribosome and stops translation by allowing recognition that a protein belongs in the ER which signals to stop translation

2. brings ribosomal component over to ER to interact with SRP receptor from the blue part which is protein end and hydrophobic that allows interaction to come together to stop translation

would you consider the SRP to be a ribozyme?

no! because it doesn’t have enzyme characteristics to speed up a reaction

how does the mechanism for SRP look like when it comes to recognizing the protein being in the ER in order to pause translation?

1. binding of SRP to signal peptide causes a pause in translation where the ER signal sequence gets recognized and binded to SRP

2. SRP-bound ribosome (signal sequence attached to SRP) ataches to SRP receptor in rough ER membrane (receptor is known to recognize and help form rough ER)

3. once binded to SRP receptor protein in rough ER membrane → translation continues and translocation begins

4. SRP and SRP receptor gets released and displaced and recycled and signal sequence gets entangled with protein translocator

what happens to the ER signal sequences for water soluble proteins following translocation?

ER signal sequences will be cleaved off by a signal peptidase following translocation

vesicular transport

• protein transport between ER, Golgi complex, plasma membrane and vesicles achieved through vesicular transport

• vesicles travel between compartments in cell along defined, regulated pathways and fuse specifically with their targets

• protein will NEVER travel across membrane

vesicular sorting depends on the assembly of a special protein coat formed at specific locations along a given donor compartment. what are those three protein coats that are involved?

1. COPII: coats from ER to golgi vesicles

2. COPI: coats from golgi to ER, golgi to plasma membrane (secretory vesicles), and within golgi

3. Clathrin: coats from plasma membrane to endosomal systems

coat proteins represent initial step in vesicle formation

• transport vesicles bud off as coated vesicles that have a distinctive cage of proteins (defined as membrane being distorted to create a "bulge” then releases to later fuse like a bubble) covering their cytosolic surface

• before the vesicle fuses with target membrane, the coat is discarded → allow two cytosolic membrane surfaces to interact directly and fuse

transport vesicles bud off as coated vesicles that have a distinctive cage of proteins covering their cytosolic surface. what are cage of proteins?

• distorts membrane to create “bulge” then releases to then later fuse into a bigger bubble

different coat proteins are involved in transport between different organelles. what are these three?

• COPII → coats ER to golgi vesicles

• COPI → coats vesicles moving from golgi to ER, golgi to plasma membrane (secretory vesicles), and within the golgi

• clathrin → transport to/from and within plasma membrane to endosomal system (endocytosis)

how does coat assembly and vesicle stability work?

• adapter proteins bind to membrane proteins and recruit coat proteins → often bind to cargo receptors - transmembrane proteins that bind soluble cargo proteins for transport

how is coat assembly controlled?

• coat recruitment GTPases control coat assembly:

  • monomeric GTPases → regulate many steps in vesicular traffic

  • Sar-1 → regulates COPII assembly

  • Arf protein → regulate COPI and clathrin assembly

Sar-1 mechanism

• Sar1-GDP = cytosol = inactive

• Sar1-GTP = ER membrane-bound = active

• active Sar1-GTP then promotes the assembly of coat complexes

• GTP hydrolysis causes coat disassembly after budding

recognition of donor vesicles by acceptor membranes is controlled mainly by two classes of proteins. what are they?

SNAREs and Rabs

SNAREs

• proteins that traps vesicle and causes fusion by snapping together of target membrane

• provide specificity

• catalyze vesicular the fusion of with target membrane

  • two kinds:

  • v-SNAREs → vesicle, thin and unfolded that threads through bubble to maintain structure, must have Rab-GTP binded

  • t-SNAREs → target membrane, within target membrane that entangles with v-SNARE to allow vesicle bubble to fuse along with target membrane where Rab-GTP lets go once vesicle fuses

Rabs

• GTP-binding protein (GTP-ases)

• recognizes to reach right membrane

• work together with other proteins to regulate initial docking and tethering of the vesicle to the target membrane

• tethering protein of Rab effector grabs or binds to Rab-GTP to allow v-SNARE and t-SNARE to entangle and allow vesicle bubble to fuse into membrane

vesicular tubular clusters

• transport vesicles leaving the ER fuse together to form intermediate compartments called vesicular tubular clusters

• clusters travel towards cis Golgi via motor proteins on microtubule tracks and generate coated vesicles going back to the ER (COPI coat) - retrograde transport

ER retrieval signals

• membrane ER resident proteins: retrieval signals in cytosolic tails, recognized by COPI coat proteins

• soluble ER resident proteins: retrieval signals within their structure, bind to receptors (ex: KDEL sequences → cannot have chemical nature altered)