MBB231 - Lecture 14: Post translational import

how does the process of post-translational import work

• free floating ribosomes build the protein before releasing it into the cytosol

• once released in the cytosol - protein either stays in the cytosol / transported into a specific organelle

• goes to either the nucleus, mitochondria, chloroplasts, or peroxisomes

what is post-translational import

where a protein is fully built in the cell’s cytosol first → then moved to its final destination

what does a protein require in order to successfully enter an organelle after being built

• a sorting signal (made from amino acids)

• a receptor - recognize the sorting signal + transports the protein to the membrane

• membrane transporter - facilitates passage into the organelle through the bilayer

what characteristic defines peroxisomes

presence of catalase defines peroxisomes → breaks down toxic hydrogen peroxide

what is the essential roles of peroxisomes

• hydrogen peroxide (H₂O₂) metabolism

  • catalase - enzyme breaks down H₂O₂ → water + oxygen

  • oxidase - enzyme oxidizes organic substances → produces hydrogen peroxide in the process

• detoxification of harmful compounds

• oxidation of fatty acids

• metabolism of nitrogen containing compounds

• catabolism of unusual substance

what are the two possible pathways that membrane lipids found in the peroxisome used to arrive there

• synthesized by peroxisomal enzymes

• transported there by lipid transfer proteins

how are most peroxisomal proteins formed

are synthesized on free cytosolic ribosomes - post-translationally imported by a C-terminal peroxisomal targeting signal

what is the mechanism of post translational protein import into the perxoisome

• newly synthesized folded protein uses its PTS-1 sequence → binds to soluble cytosolic import receptor (Pex5)

• complex moves to the peroxisomal membrane → interacts with the receptor (Pex14) at the membrane

• protein dissociates from Pex5 → moves across the membrane to peroxisomal matrix

• once cargo is released - Pex5 stays embedded in the membrane → tagged with Ubiquitin (Ub) by Pex 2, Pex10, Pex12

• ubiquitinated Pex5 is extracted from the membrane → returned to the cytosol to be reused

  • step requires energy from ATP hydrolysis → carried out by AAA-ATPASE complex ( Pex1 & Pex 6)

what are the 4 sub compartments in the mitochondria in which proteins can be delivered to

• outer mitochondrial membrane

  • relatively permeable

  • has porins → allows movement of small molecules + ions across the outer membrane

• inner mitochondrial membrane

  • highly impermeable

  • acts as a permeability barrier to most solutes

• matrix

  • in the inner membrane

  • contains enzymes with mitochondrial function

  • contains DNA + ribosomes

• intermembrane space

  • area between the inner / outer mitochondrial membrane

  • intracristal space - folds in the inner membrane → regions where protons accumulate during electron transport process

proteins that enter the mitochondria must be in what state and contain what

must be in an unfolded state + contain pre-sequence (matrix) or internal targeting sequence (inner membrane)

unfolded state → need to be in an unfolded + linear state to fit through the narrow entry channels

pre-sequence → directs the protein to the matrix

internal targeting sequence → directs the protein to the inner membrane

how can translocation across both membranes occur at the same time

can occur at sites where the inner + outer membrane are close together

what are the two mitochondrial transport complexes used to pull proteins across the inner/outer mitochondrial membranes

translocase of the outer membrane (TOM) - transport channel for the outer membrane

translocase of the inner membrane (TIM) - transport channel for the inner membrane

how do proteins travel from the cell’s cytosol → matrix of the mitochondria

• cytosolic proteins are bound by chaperones → keeping them in an unfolded state

• pre-sequence is formed by N-terminal alpha-helix → sequence binds to the receptor component of TOM (outer mitochondrial membrane) → sequence + cytosolic protein is positioned into the TOM channel

  • pre-sequence contains positively charged + hydrophobic amino acids

• transport of proteins to the matrix → TOM and TIM23 channels are brought together

• movement into matrix is powered by electrical potential (matrix - negative / pre-sequence - positive)

• two hypothesis of how mitochondrial proteins are pulled into matrix

  • chaperones use energy from ATP hydrolysis → to pull and bring the peptide into the matrix

  • random diffusion allows the peptide to poke the N-terminus through the channel → becomes bound by a chaperone → continues to diffuse and latch on chaperones (to prevent backsliding) until it reaches the matrix

• once the protein is in the matrix → pre-sequence is cleaved by peptidase → protein folds into its native conformation

what are the three energy dependent steps required to import a precursor protein into the mitochondrial matrix

• cytosolic ATP hydrolysis

  • cytosolic Hsp70 chaperone protein keeps the newly synthesized protein in an unfolded state

  • ATP hydrolysis releases cytosolic Hsp70 from the polypeptide chain

  • allows the positively charged N-terminal pre-sequence to insert into the TOM complex (outer membrane) + TIM23 complex (inner membrane)

• membrane potential across the inner membrane

  • positively charged pre-sequence is driven across the inner mitochondrial membrane → matrix

  • electrochemical gradient of hydrogen established by the electron transport chain → leaves the matrix side negatively charged (compared to the intermembrane space)

  • electrophoretic forces pulls the positively charged sequence through the TIM23 channel → matrix

• mitochondrial ATP hydrolysis

  • as the polypeptide chain becomes exposes into the matrix space → mitochondrial Hsp70 binds to the regions on the polypeptide chain

  • is ATP dependent as ATP hydrolysis drives a conformation change → actively pulls the chain into the matrix and prevents it from sliding backward

how are targeted proteins brought into the inner mitochondrial membrane

• cytosolic proteins are bound to chaperones → keeps them unfolded

• pre-sequence (formed by the N-terminal alpha helix) binds to the receptor component of TOM

• positioned into the TOM channel on the outer mitochondrial membrane

• TOM and TIM 22 channels are brought together

• internal targeting sequence (hydrophobic amino acid sequence) → signals the TIM22 channel to allow the protein to pass the lipid bilayer

why is an impermeable membrane essential for the generation of ATP

acts as a sealed barrier that allows the cell to build and maintain a proton electrochemical gradient

what is the process of oxidative phosphorylation (where cells generate energy - ATP)

• high energy electrons travel through protein complex embedded in the inner mitochondrial membrane

• electrons move - releases energy → proteins use this energy to pump protons out from the matrix

• oxygen uses the used electrons and combines them with protons → forms water

• since hydrogen is being accumulated outside of the matrix - some come back into the matrix through ATP synthase (proton channel)

• enzyme use the energy of hydrogen returning back into the matrix to attach an inorganic phosphate + ADP → ATP

what do abolishing agents such as DNP do to ATP synthesis

• stops ATP synthesis without blocking the uptake of oxygen

• electron transport and H+ pumping continues → no proton gradient is generated

• DNP can diffuse through the inner mitochondrial membrane and carry protons ( can continuously do it )

  • allows protons to flow back into the matrix without having to pass through ATP synthase

  • enzyme does not have any energy (potential energy) to use to formulate ATP from ADP + Pi

how are proteins made outside of the chloroplast shipped inside + sorted inside the chloroplast

crossing into the chloroplast:

• helper proteins (Hsp70/Hsp90) keep the protein unfolded

• protein passes through Toc complex (outer membrane) & Tic complex (inner membrane)

• once inside the stroma (main inner fluid) - enzyme (peptidase) snips off the stroma targeting domain tag

sorting to final destination: 3 pathways

• stay in the stroma

  • protein is folded into its active 3D shape by Hsp60 - stays in stroma

• directed to thylakoid lumen (inside of thylakoid)

  • protein has a thylakoid transfer domain - cross the thylakoid membrane into the lumen

  • thylakoid transfer domain tag is snipped off by enzyme

• embedded on the thylakoid membrane

  • some proteins can be inserted on to the thylakoid membrane

  • by co-translationally being inserted

what two membranes consist of the nuclear envelope

• inner membrane - defines the nucleus

• outer membrane - contiguous with the ER

what do nuclear pores do for the nucleus

it fuses the two membranes (inner and outer) - forms sites for transport between the cytoplasm - nucleoplasm

what molecules move from cytosol → nucleus and nucleus → cytosol

cytosol → nucleus

• histones

• DNA & RNA polymerase

• transcription factors

• RNA - processing proteins

• ribosomal proteins

nucleus → cytosol

• tRNA

• mRNA

• RNP complexes

how do small molecules travel through the nuclear pores through passive transport

• small particles (<10nm in diameter) pass through pores at a rate proportional to the size of the particle

• nuclear pore complex (NPC) contains aqueous diffusion channels → small particles can travel through

• proteins up to 40kDa (approx. 5nM) can use passive transport through the pores

how do large proteins + RNA use active transport through the nuclear pores

• large particles are actively transported across the membrane

• nuclear localization signals (NLS) & nuclear export signals (NES) allow proteins to be recognized + transported by the nuclear pore complex

how does the cell maintain a Ran-GTP gradient

• in the nucleus - high Ran-GTP

  • helper protein attached to chromatin (Ran-GEF) swaps GDP → GTP

  • turns Ran → Ran-GTP

• crossing the nuclear pore

  • ran moves back and forth between nucleus + cytosol through the nuclear pores

• in the cytosol - high Ran-GDP

  • helper protein in the cytosol (Ran-GAP) triggers Ran to break down from GTP → GDP (releases an inorganic phosphate)

  • turns Ran → Ran-GDP - keeping Ran-GTP levels low and Ran-GDP levels high in the cytosol

what does Ran - GTP/GDP do

regulates nuclear import and export

explain how nuclear import occurs with Ran (molecular switch) and importin (escort protein)

• cytoplasmic protein with a nuclear localization signal (NLS) - recognized by a family of nuclear import receptors (importins)

• importins binds to the NLS and mediates the movement of the protein to a nuclear pore

• docks with the nuclear pore complex - transported into the nucleus

• inside the nucleus - importin associates with Ran (GTP binding protein) → causes importin to release the protein with the NLS

• Ran-GTP-importin complex is transported back to the cytoplasm through the NPC

• in the cytoplasm - NPC is released & GTP is hydrolyzed

• Ran-GDP travels back to the nucleus through NPC → encounters GAP - triggers conversion of Ran-GTP → Ran-GDP

how are ribosomes exported out of the nucleus and into the cytoplasm

• ribosomes are assembled in the nucleolus - where ribosomal RNA (rRNA) is combined with proteins to make a small (pre-40s) and a large (pre-60) ribosomal subunits

• ribosomal subunits are individually exported from nucleus → cytoplasm through the nuclear pore complex (NPC)

• export is mediated by export receptors ( Crm1 / exportin 1) + Ran-GTP

how is RNA exported out of the nucleus and into the cytoplasm

miRNA / tRNA - bind to nuclear export receptors → uses Ran-GTP gradient

• have a nuclear export sequence (NES) - binds to nuclear export receptors

• Ran-GTP grabs onto the cargo inside the nucleus → carries it out to the cytoplasm

• hydrolysis of Ran-GTP - releases the miRNA/tRNA into the cytoplasm

mRNA - uses a different system