Part 2.2: Transport to Organelles and Nucleus

Mitochondrial Import: Background

• most mitochondrial proteins are encoded in the nucleus and synthesized in the cytosol

  • precursor proteins are synthesized on cytosolic ribosomes; chaperons like Hsc70 bind to them and keep them unfolded

  • only unfolded protein can thread thru the import pores

• mitochondrial precursor proteins have an N-terminal targeting signal that is different from ER signal sequences

  • typically amphipathic ⍺-helix

  • recognition depends on specific protein interactions with mitochondrial receptors, not just hydrophobicity

• ATP hydrolysis helps drive import into the matrix

Mitochondrial Transport: TOM

Translocator of Outer Membrane Complex

• TOM20: receptor recognizing the targeting signal (import receptor)

• TOM40: the actual pore that allows the unfolded protein to cross the outer membrane

• After TOM20 binds the signal, the precursor is handed off to TOM40

Mitochondrial Transport: TIM

Translocator of Inner Membrane Complex

• works with TOM at contact sites where the inner and outer membranes are close

• translocates proteins across the inner membrane into the matrix

Mitochondrial Transport: Driving Forces

• matrix chaperones (eg. Hsc70) bind to the incoming protein inside the matrix and use ATP hydrolysis to pull it through

• proton motive force (PMF) across the inner membrane also helps drive import, especially for inner membrane proteins

  • the intermembrane space is more positively charged relative to he matrix, the PMF pulls + charged portions of precursors inward

Mitochondrial Matrix: Additional Steps

• after import, the N-terminal targeting signal is cleaved by a peptidase

• final folding is assisted by matrix chaperones

Mitochondrial Matrix: Steps

• protein synthesized in cytosol → bound by cytosolic chaperones

• N-terminal mitochondrial signal recognized by TOM 20 receptor

• protein enters TOM40 pore

• protein passes TIM complex at inner membrane contact sites (only for matrix proteins)

• matrix chaperones + ATP pull protein in; PMF may assist

• signal peptide removed → protein folds into functional form

Mitochondrial Import: Figure

Mitocondrial Import: Porins

• the TOM complex cannot alone integrate β-barrel porins into the lipid bilayer from outside

• porins are first transported into the intermembrane space, binding specialized chaperones

• porins then bind to the SAM complex in the outer membrane, which inserts them into the outer membrane

• the central subunits of the SAM complex are homologous to a bacterial outer membrane protein that helps insert β-barrel proteins (eg. BAM in bacteria)

Chloroplast Import: Background

• protein import into chloroplasts happens post-translationally (proteins are synthesized

  • resembles transport into mitochondria

• proteins remain unfolded, assisted by cytosolic chaperones, until they reach the chloroplast

• signal sequences for import into chloroplasts resemble those for mitochondria

Chloroplast Import

• TOC (translocon at outer chloroplast membrane)

• TIC (translocon at inner chloroplast membrane)

  • these are homologous to mitochondrial TOM and TIM complexes

• the chloroplast-targeting signal sequence at the N-terminus directs the protein to the TOC receptor

Chloroplast Import: Thylakoid

• chloroplasts have an additional membrane-enclosed compartment, the thylakoid

  • the photosynthetic system is located in the thylakoid membrane

• proteins destined for the thylakoid need 2 targeting signals

  • chloroplast signal sequence (for import into stroma)

  • thylakoid signal sequence (for further transport across thylakoid membrane)

Chloroplast Import: Two-Step Transport Pathway

• precursor proteins cross the outer and inner envelope membranes thru the TOC and TIC complexes at contact sites

  • once inside the chloroplast-targeting signal is cleaved off

• the remaining thylakoid signal sequence directs the protein to the thylakoid membrane or lumen

  • via a similar system to SRP and SecYEG of bacteria

Peroxisomes

• all eukaryotic cells have peroxisomes

• sites of oxygen utilization, β-oxidation of fatty acids into acetyl-CoA, ethanol oxidation to acetaldehyde

• surrounded by a single membrane

• acquire proteins from the cytosol, including oxidative enzymes, such as catalase and urate oxidase (present at high concentrations in the peroxisome)

  • enzymes are not made inside

Peroxisomal Targeting Signal (PTS1)

• the sequence (Ser, Lys, Leu) at the C-terminus of peroxisomal proteins is the import signal

• not cleaved off after import

Peroxisome Protein Import: Peroxins

• at least 23 PEX proteins mediate peroxisomal import

• import is post-translational: proteins are fully folded in cytosol before import

• PEX5: the cytosolic receptor for the SKL signal, binds SKL-containing proteins in the cytosol, docks at the peroxisome membrane

  • after delivering cargo, PEX5 is ubiquitinated, extracted and reuse

• PEX13 & PEX14: form the docking complex on the peroxisome membrane, accept PEX5+cargo

• PEX2, PEX10, PEX12: required for the translocation of cargo and for recycling PEX5 back to the cytosol

Peroximal Protein Import FIGURE

Peroxisomal Import: Zellweger Syndrome

• caused by a defect in importing proteins into peroxisomes and peroxin function

• individuals have cells with empty peroxisomes, leading to severe brain abnormalities and die soon after birth

Primary Hyperoxaluria

• protein targeting is essential, proteins must be delivered to exactly the right organelle to function

• Normal situation: the enzyme alanine:glyoxylate aminotransferase (AGT) normally needs to be in the peroxisome

  • contains a PTS1 at its C-terminus

  • in peroxisomes, AGT convert glyoxylate → glycine, preventing toxic buildup

• Disease situation: PTS1 signal is altered, now interpreted as mitochondrial targeting signal

  • AGT cannot access glyoxylate, glyoxylate accumulates and converted to oxalate

  • oxalate forms insoluble calcium oxalate crystals leading to kidney stones in early childhood and renal failure if untreated

PEX13 Dodecamer Ring: Amphipathic Helix

• PEX13 contains a long amphipathic helix, and 12 copies of this helix assemble into a ring-like pore in the peroxisomal membrane

• the helices tilt and alternate orientation

  • the downstream TMS and SH3 domains also alternate around the ring (black dotted lines)

• the ring creates a large central opening

PEx13 Dodecamer Ring: Hydrophobicity/Hydrophilicity 

• hydrophobic residues of each helix face outward and interact with the lipid bilayer

• hydrophilic residues face inward, forming a water-filled channel

PEx13 Dodecamer Ring: In the Membrane

• PEX13 had a Tyr/Gly (YG) domain

• inside, the YG domains from each subunit project inward to form a dense meshwork that acts as a selective barrier for protein import

  • nuclear pore-like hydrogel

  • the meshwork is held together by cohesive interactions between the Y residues of the YG domain

• PEX5 partitions into this meshwork by transiently disrupting the cohesive interactions using its WxxF/Y motifs

  • these motifs temporarily disrupt the YG gel, allowing PEX5+cargo to pass thru the pore, then the gel reseals behind them

PEx13 Dodecamer Ring: Experimental Evidence

• the purified YG domain of PEX13 forms a hydrogel

• a concentrated solution (40 mg/mL) of the protein was pipetted into silicone tubing and allowed to gel, then squeezed out onto a colored surface and photographed

  • gelation was observed with the WT protein, but a mutant (Tyr→Ser) remained fluid

  • therefore, tyrosines are essential for hydrogel formation

PEX5 Receptor

• cytosolic receptor that recognizes cargo that contains SKL signal and guides it through a docking complex on the peroxisomal membrane.

  • after releasing the proteins inside, the PEX5 receptor is recycled back to the cytoplasm to pick up more cargo

• PEX5 is rich in aromatic residues (Trp, Phe, Tyr) in its N-terminal region, specifically within its repeated WxxF/Y motifs

  • allow for aromatic-aromatic (π-π) stacking interactions, which allow PEX5’s motifs to disrupt or slip between the Tyr’s in the YG domain of PEX13

  • allows to carry cargo thru the pore and exit again into the cytosol once cargo is released

PEX5 Receptor: Experiment

• YG-hydrogel droplets (40mg/ml) were prepared in glass-bottomed dishes; permeation of the gels by fluorescently labeled PEX5 or other proteins was images by point-scanning confocal microscopy

• scheme depicting the PEX5 fragments that were fused to GFP. PEX5’s N-terminal region contains several WxxxF/Y motifs

• YG-hydrogel droplets were bathed in buffer containing the indicated GFP-fusion proteins (or GFP alone), and the interface between the buffer and gel was imaged over time

  • shown at 3 selected time points; the fold enrichment of each protein, relative to buffer, across the imaged field is plotted below (mean ± the range of three experiments).

PEX Transport Cycle: Step 1

PEX5 recognizes and binds to cargo proteins in the cytosol that contain an SKL signal

PEX Transport Cycle: Step 2

Cargo-bound PEX5 is recruited to peroxisomes by a complex containing the membrane proteins PEX13 and PEX14

PEX Transport Cycle: Step 3

Cargo-bound PEX5 traverses the membrane through a conduit formed by multiple copies of PEX13. The conduit contains a dense meshwork formed from the PEX13 Tyr/Gly- rich domain, into which PEX5 partitions using its WxxF/Y motifs

PEX Transport Cycle: Step 4

PEX5 is drawn into the matrix by favourable lumenal interactions between the receptor WxxxF/Y motifs and PEX14 oligomers.

• the lumenal interactions are energetically favorable, so they pull PEX5 deeper into the matrix, bring the cargo with it

PEX Transport Cycle: Step 5

• PEX5 now needs to be removed from the matrix 

PEX5 spools its flexible N-terminus into the cytosol through a pore in the PEX2-PEX10-PEX12 ubiquitin ligase complex, which then monoubiquitinates the receptor on a conserved cysteine

PEX Transport Cycle: Step 6

Monoubiquitinated PEX5 is pulled out of the matrix thru the ligase pore by the PEX1-PEX6 AAA ATPase, which unfolds the receptor and causes cargo to be stripped off inside the matrix

• energy dependent step

PEX Transport Cycle: Step 7

PEX5 refolds in the cytosol and ubiquitin is removed by de-ubiquitinases resetting the receptor for another import cycle

PEX Transport Cycle Figure

Nuclear Envelope

• encloses the DNA and defines the nuclear compartment

• the envelope consists of two membranes, penetrated by nuclear pore complexes

• the inner membrane is an anchoring site for chromatin and for the nuclear lamina

• the outer nuclear membrane is continuous with the membrane of the ER

• protein traffic occurs continuously between the cytosol and the nucleus 

  • many proteins synthesized in cytosol function in the nucleus (eg. histones, DNA and RNA polymerases, gene regulatory proteins and RNA processing proteins)

• tRNAs and mRNAs synthesized in the nuclear compartment are exported to the cytosol

ER

• organized into a netlike labyrinth of branching tubules and flattened sacs

• the rough ER has many ribosomes bound to its cytosolic surface, and is a major site or protein production

• proteins are transported into the ER as they are synthesized

• the ER membrane is the site of production of all the transmembrane proteins

  • also produces most of the lipid for the rest of the cell

• almost all secreted proteins to the cell exterior are initially delivered to the ER lumen

• stores Ca²⁺ ions

• in liver cells, the ER has a surface area 25x that of the plasma membrane

Smooth ER

• the transitional ER is a smooth ER from which transport vesicles are budding off

• in cells that specialize in lipid metabolism, the smooth ER is abundant

• the ER store enzymes (cytochrome P450 family) that metabolize lipid-soluble drugs (rendered water soluble to leave the cell and be excreted in the urine)

Nuclear Envelope/ER Figure

Nuclear Import

• nuclear import requires NLS + importin (nuclear transport receptor) + Ran-GTP cycle (energy) to get through

• Nuclear pore complexes perforate the nuclear envelope

  • some proteins continually shuttle back and forth between the nucleus and the cytosol

  • the relative rates of their import and export determine the steady-state localization

Nuclear Pore Complex (NPC): Structure

• selective hydrogel-like barrier formed by FG repeats (Phe-Glyc) 

• 3000-4000 NPCs per cell

• built from ~30 different proteins (nucleoporins)

• unlike PEX pores, the NPC isn’t a simple membrane channel, it sits outside the bilayer like a giant ring complex

NPCs: FG-Fibrils

• the pore is gated by FG-fibrils

• FG= Phe-Gly repeats, which form unstructured, hydrophobic tendrils inside the pore (a hydrogel-like mesh)

• the mesh blocks passive diffusion of large molecules (>60 kDa)

  • ribosomes are ~30nm in diameter and thus cannot diffuse thru the NPC

• only proteins with the right importins can get through

  • Nuclear transporter receptors (NTRs) diffuse thru the FG meshwork by transiently binding to F residues using hydrophobic patches

NPCs: How transport works

• importins have hydrophobic patches and are specialized for the transport of a subset of cargo proteins

• importins bind the NLS and bind Phe in FG repeats transiently

  • hop from FG-repeat to FG-repeat

  • this allows selective passage thru the mesh

NLS

• nuclear localization signals are responsible for the nuclear import process

• short Lys-Arg rich positive sequence

• signal is located anywhere in the protein (loops or patches on protein surface), the precise location of the signal within the protein is not important

• fully folded proteins can be transported into the nucleus thru an NPC

NPC Figure

Nuclear Import Transport model: Import

• cargo binds importin in the cytosol (importins recognize NLS sequences)

• importin-cargo moves thru the FG-mesh of the NPC

• Ran-GTP binds to the importin on the nuclear side of the pore

• Ran-GTP binding causes the receptors to release their cargo

• the importin with Ran-GTP is transported back to the cytosol

• Ran-GAP in the cytosol triggers the hydrolysis of GTP, thereby converting it to Ran-GDP

• the import receptor is then ready for another cycle of nuclear import

Nuclear Import Transport model: Export

• exportins binds both cargo and Ran-GTP inside the nucleus (export cargos typically have nuclear export signals)

• exportin-cargo-Ran-GTP passes thru the pore

• in the cytosol, Ran-GTP hydrolyzes GTP → GDP

• the export-receptor releases both its cargo and Ran-GDP in the cytosol

• free export receptors are then returned to the nucleus to complete the cycle

Nuclear Import Transport: Gradient

• the import cycle is powered by a Ran-GTP gradient across the pore

• the gradient arises because of the exclusive nuclear localiztion of proteins called Ran GEFs

  • Ran-GEF is only in the nucleus, GEF=guanine exchange factor → loads GTP onto Ran to produce Ran-GTP

• these proteins exchange GDP for GTP on Ran molecules

• there is an elevated RanGTP concetration in the nucleus compared to the cytoplasm

• Ran-GAP is only in the cytosol

  • GAP= GTPase activating protein → hydrolyzes Ran-GTP → Ran GDP

Nuclear Import Transport Model Figure

Ran Cycling Mechanism

• translocation thru the pore is not energy-dependent; the cargo passes thru the pore with the assistance of importins 

• the whole import cycle and directionality requires Ran-GTP hydrolysis (but not used energy to push cargo physically, rather it resets the receptors)

• the driving force for transport depends on the gradient of Ran-GTP

• the gradient of Ran-GTP is made because of the preferential location of Ran-DEG and Ran-GAP

Ran Cycling Mechanism: Regulatory Proteins

• Ran-GEF (nuclear) converts Ran-GDP → Ran-GTP

  • a GTP exchange factor

• Ran-GAP (cytosolic) converts Ran-GTP→Ran-GDP

  • a GTPase-activating protein

Ran Cycling Mechanism:

The Piggy-Back Transport Model

• the cargo itself doesn’t need a signal if it associates with signal-bearing partner