MCP 1-11 Flashcards
Transport via Protein Translocators
Directly transport proteins from cytosol –> organelle
- Co-translationally for cytosol –> ER
- Post-translationally for cytosol –> mitochondria and peroxisomes
Mechanisms of protein import into organelles
- through nuclear pores
- across membranes
- by vesicles (i.e. ER -> Golgi, Golgi -> PM, Golgi -> lysosomes
Signal Sequences
Purpose: direct proteins to the correct organelles
- stretch of 3-60 aa within protein
- may be removed by signal peptidase after transport (N terminus)
- different types specify different locations
- functionally interchangeable
- recognized by specific receptors
Function of Mitochondria
- provide ~90% of cell’s energy (ATP)
- near sites of high ATP use
- more in cells with higher energy demands
Structure of Mitochondria (4 compartments)
Two compartments separated by two membranes:
- Matrix space: enzymes for beta-oxidation (break down FA) and TCA cycle; location of mito. DNA genome and transcription/translation machinery for mito.genes
- Inner membrane: cristae increases surface area, ETC, ATP synthase, transport proteins, H+ electrochemical gradient (drives ATP synthesis)
- Outer membrane: porin forms channels
- Intermembrane space: between inner/out membranes, cytochrome c
Role of Mitochondria in Apoptosis
Cytochrome c released from intermembrane space into cytosol –> caspase cascade (proteolytic cascade, cleaves key cellular proteins)
Transport of proteins into mitochondria
Protein binds TOM (outer membrane) -> moves laterally until it hits TIM complex (inner membrane) -> protein moves across, into matrix -> signal sequence cleaved by mitochondrial signal peptidase -> chaperone proteins fold into final conformation
* Energy requirements: ATP gradient, electrochemical gradient across inner membrane
Features of mitochondrial genome
- very small circular dsDNA
- encodes 2 rRNAs, 22 tRNAs, 13 mRNAs
- little regulatory sequence
- no introns
- genetic code is slightly different (4 codons have different “meanings” from codons in nuclear genome)
- ~10-20 copies of genome/mitochondrion
Replication of Mitochondrial DNA
- occurs throughout cell cycle, not limited to S-phase like nuclear DNA
- mtDNAs chosen at random to replicate
- total # mtDNA doubles in every cell cycle
- Origin of replication on each strand
Transcription of Mitochondrial DNA
- both strands of DNA transcribed from single promoter region on each (HSP = heavy strand promoter, LSP = light strand promoter)
- produces 2 giant RNAs, each a transcript of one full-length DNA strand
- Each RNA cleaved into 2 rRNAs, 22 tRNAs, and 13 mRNAs
Translation of Mitochondrial mRNAs
- occurs in matrix
- uses tRNAs and rRNA encoded in mtDNA (mt genome)
- only produces 13 polypeptides (encoded in the 13 mRNAs), all are subunits involved in ET and ox. phosphorylation
Functions of Peroxisomes
- Oxidative degradation (use oxygen to oxidize –> hydrogen peroxide), catalse driven reactions (convert left over hydrogen peroxide to water and oxygen)
- Beta oxidation (very long chain fatty acids that can’t be broken down by mito. –> acetyl CoA)
- Synthesis of cholesterol, bile acids, and some lipids (ex. plasmalogen synthesis for myelin sheaths)
Disorders of peroxisome biogenesis
- defects in proteins required for biogenesis
- lack many peroxisomal enzymes or peroxisomes are absent from cells
Zellweger Syndrome
- disorder of peroxisome biogenesis
- peroxisomal enzymes synthesized normally but not imported
- empty peroxisomes
- lethal in early infancy
Deficiency of single peroxisomal enzyme
- defect in synthesis, import or function of one peroxisomal protein
- less severe phenotype
- partially functional peroxisomes
X-linked Adrenoleukodystrophy (ALD)
- deficiency of a single peroxisomal protein
- peroxisomes lack membrane protein involved in degradation of very long chain FA -> build up -> leads to demyelination of neurons, dysfunction of nervous system, and adrenal insufficiency
- lethal in mid childhood
Treatment of ALD
- Allogeneic stem cell transplant: high morbidity, compatible donor cells not always available, must be performed at early stage of brain lesions
- Gene therapy: recent success in two patients
Gene therapy for ALD
- Hematopoietic stem cells (HSCs) collected from two 7 y.o ALD pt
- HSCs corrected ex-vivo using HIV-derived lentiviral vector expressing wt ALD protein
- Chemotherapy used to eradicate bone marrow, pt own corrected HSCs were infused
- Progeny of HSCs distribute throughout body, including brain microglia responsible for maintaining myelin
- 4 year follow up: 10-11% of hemo. cell lines stably expressed wt protein, results similar to allogeneic stem cell transplant
- First successful clinical test of HIV-derived vector in HSC-based gene therapy*
