peroxisomes, biogenesis and function, posttranslational import of proteins into organelles

nuclear export of mRNA

• splicing factors bound to unspliced introns contain nuclear retention signal (NRS)

  • once introns are spliced out, mRNA no longer has NRS attached

• addition of poly(A) tail prepares transcript for export

• some RNA-binding proteins are removed in the nucleus and remain in the nucleus (ex: hnRNP C1)

• some RNA-binding proteins are removed in the cytoplasm and transported back into the nucleus (ex: hnRNP A1)

• hnRNPs exchanged for mRNPs in cytoplasm, result is mRNA transcript bound to mRNPs

UAP56

• ATP-dependent RNA helicase

• concentrated in speckles

• acts as a molecular switch for human mRNA export from the nucleus

• uses ATP to remodel mRNA complex (mRNA + proteins) to allow docking in the nuclear basket of the NPC

UAP56 mechanism

1. mRNA complex (mRNA + proteins) is recognized by THO, which deposits UAP56 onto mRNA

2. transcription-export complex (TREX) binds mRNA

3. with the removal of THO and addition of ATP, UAP56 binds mRNA and remodels it so it can bind TREX-2 in the nuclear basket of the NPC

4. UAP56 detaches and, with the help of NXF1 (TAP) and NXT1 (p15), mRNA is exported through the NPC

nuclear export of unspliced or partially spliced HIV-1 mRNA

• unspliced (or partially spliced) viral mRNA contains Rev response element (RRE) and splicing factors containing a nuclear retention signal (NRS)

• Rev binds to the mRNA at the RRE and is recognized by Crm1

• whole complex is exported by Crm1, regardless of NRS on splicing factors

functions of importin-α unrelated to nuclear trafficking

• nuclear envelope and membrane fusion

• regulation of transcription

• stress response - stress granules

• regulation of cell differentiation

• cell scaling

• chaperone function

functions of importin-β family members unrelated to nuclear trafficking

• mitosis

• stress response - stress granules

• protein disaggregation

• chaperone function

• protein translation at the synapse

Ran function unrelated to nuclear trafficking

mitosis

CAS function unrelated to nuclear trafficking

regulation of transcription

Crm1 functions unrelated to nuclear trafficking

• transport through the nucleolus

• promotes Nup solubility

• modulates biocondensate formation

Nup98 function unrelated to nuclear trafficking

control of transcript stability

DDX3 (RNA helicase) function unrelated to nuclear trafficking

stress response - stress granules

formation of gel-like or glass-like condensates

• relevant to certain diseases (neurodegeneration, cancer, viral infections)

• caused by misfolded proteins or mutations in FUS or TDP43

• can become permanent

stress granules

• accumulations of RNA and proteins (especially RNA-binding proteins) during stress events to protect them

  • protect RNA from degradation

• generated if stress is not lethal

• contains importin-α1

• sequesters some apoptotic factors (factors that cause apoptosis) to prevent apoptosis from occurring

droplet compartments

• mixing of proteins and RNA can create droplets

• addition of more RNA can produce hollow droplets

importance of stress granules in human disease

• tumour microenvironment and chemotherapy induce stress granule assembly and creates tumour resistance

  • to increase treatment effectiveness, need to prevent formation of stress granules

• persistent granules in neurons lead to neurodegeneration

• impairment of stress granule formation by certain viruses leads to enhanced infectivity

TDP-43 and FUS

• RNA-binding proteins involved in several aspects of RNA homeostasis (splicing, mRNA export from nucleus)

• mutations have been associated with ALS (amyotrophic lateral sclerosis) and FTD (fronto-temporal dementia)

• mutants are often prone to aggregation

limit of pathological LLPS during neurodegeneration by nuclear transport factors

• shuttling of proteins prone to aggregating (chaperone-like function)

• help disassemble pre-existing protein aggregates

peroxisomes

• single membrane surrounds perisomal matrix

• play a role in many detoxification reactions

  • catalase enzyme oxidizes different compounds and removes H₂O₂ → control of cellular redox status

• β-oxidation of long chain fatty acids

• involved in the synthesis of plasmalogens (→ myelin)

biogenesis of peroxisomes

• from scratch: vesicles derived from the ER and mitochondria fuse to form pre-peroxisomes, then mature into peroxisomes

• division: pre-existing peroxisomes replicate by growth and divide into new peroxisomes

factors required for protein import into the peroxisomal or mitochondrial matrix

• signals

• energy

• cellular import apparatus

peroxin

peroxisome-related protein, involved in targeting of proteins to the peroxisomal matrix or membrane

protein transport to peroxisomes

• all peroxisome proteins are encoded by nuclear genes

• protein import is posttranslational

• folded proteins can be imported into the matrix

• protein import requires ATP

• peroxins mediate peroxisomal biogenesis and protein import

• protein import into the matrix is mediated by a transient translocon and involves phase separation at the translocon

targeting signals for peroxisomal matrix

• PTS-1

  • SKL sequence

  • always located at C-terminus

  • never cleaved off

• PTS-2

  • some are cleaved off

  • less common than PTS-2

targeting of PTS-1-containing proteins to the peroxisomal matrix

1. protein folds

2. SKL signal binds SKL-receptor (PEX5)

3. complex docks at membrane proteins

4. complex moves into the matrix and disassembles

5. SKL-receptor returns to cytoplasm

protein import into peroxisomes

• membrane protein PEX13 contains conserved YG region (YG repeats) that forms a meshwork in the lipid bilayer

• PEX5 partitions into the YG domain meshwork

• PEX5 moves cargo across the meshwork into peroxisomal lumen

recycling of PEX5

1. dissociation of cargo from PEX5 in peroxisomal lumen

2. PEX5 modified by PEX2-10-12 (membrane-bound complex) and becomes ubiquitinated

3. ubiquitinated PEX5 in complex recognized by PEX1 and PEX6 (AAA ATPase) and pulled into cytoplasm

4. Ub removed from PEX5 in cytoplasm, PEX5 can be reused

peroxisome biogenesis disorders

• Zellweger syndrome

• neonatal adrenoleukodystrophy

• infantile Refsum disease

• Rhizomelic chondrodysplasia punctata

single peroxisomal protein defects

• X-linked adrenoleukodystrophy

• hyperoxaluria type I

• Refsum disease

• thiolase deficiency

Zellweger syndrome

• mutation in PEX5

• affects peroxisomal import of all proteins with PTS sequence

hyperoxaluria type I

• PTS-2 signal mutated → MTS

• single protein ends up in mitochondria instead of peroxisome

• patients prone to producing kidney stones

PEX5

PTS-1 receptor (SKL sequences)

PEX7

PTS-2 receptor

forms of energy in mitochondria

• ATP

• electrochemical gradient across inner membrane

targeting to mitochondrial matrix

• protein contains mitochondrial targeting sequence (MTS) at N-terminal end

• proteins have to be unfolded

• MTS forms an amphipathic helix (positive on one side, hydrophobic on other)

• MTS removed during trafficking

mitochondrial protein translocators

• TOM complex (translocase of the outer membrane)

• TIM23 complex (translocase of the inner membrane)

• TIM22 complex (translocase of the inner membrane)

• OXA complex (oxidase assembly protein, inner membrane)

mitochondrial transport factors (other than translocators)

• sorting and assembly machinery (SAM, outer membrane)

• presequence translocase-associated motor (PAM, associated with TI 23)

• mitochondrial processing peptidase (in cytosol, cleaves off MTS)

energy requirements from protein sorting to the mitochondrial matrix

• ATP in cytosol for cytosolic Hsp70s to help unfold proteins

• membrane potential to facilitate entry of protein into TIM23 complex (through inner membrane)

• ATP in matrix to cause conformational change of Hsp70 and for mitochondrial Hsp70, Hsp60, and Hsp10 to help unfold proteins

protein sorting to inner mitochondrial membrane (single-pass)

• protein enters matrix through TOM and TIM23 complexes

• signal sequence is cleaved off as protein exits TIM23 complex

• second signal sequence binds OXA complex in inner membrane

• OXA complex helps protein properly insert into membrane

• protein synthesized in the mitochondria only needs second signal sequence and binds to OXA right away

• energy needs: ATP in cytosol (only if protein is from cytosol), electrochemical gradient, ATP in matrix

protein sorting to inner mitochondrial membrane (multi-pass)

• protein synthesized in cytosol has internal MTS

• tiny TIMS (intermembrane space chaperones) bind protein and shield hydrophobic regions, help insert protein into TIM22 complex

• TIM22 complex helps protein insert properly into membrane (with multiple membrane-spanning domains)

• energy needs: ATP in cytosol, electrochemical gradient, energy to insert properly into membrane

protein sorting to outer mitochondrial membrane

• protein enters intermembrane space through TOM complex

• tiny TIM chaperones help protein enter SAM complex

• SAM complex helps protein fold properly and insert into membrane

• energy needs: ATP in cytoplasm

human deafness dystonia syndrome

• X-linked recessive disorder

• results from loss-of-function mutation in the nuclear-encoded deafness dystonia peptide 1 (DDP1)/translocase of mitochondrial inner membrane 8A (TIMM8A)

• characterized by hearing loss early in life, problems with movement, impaired vision, and behaviour problems