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Mechanisms of protein import into organelles

1. through nuclear pores
2. across membranes
3. by vesicles (i.e. ER -> Golgi, Golgi -> PM, Golgi -> lysosomes

1

Transport via Protein Translocators

Directly transport proteins from cytosol --> organelle
- Co-translationally for cytosol --> ER
- Post-translationally for cytosol --> mitochondria and peroxisomes

2

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

3

Function of Mitochondria

- provide ~90% of cell's energy (ATP)
- near sites of high ATP use
- more in cells with higher energy demands

4

Structure of Mitochondria (4 compartments)

Two compartments separated by two membranes:
1. Matrix space: enzymes for beta-oxidation (break down FA) and TCA cycle; location of mito. DNA genome and transcription/translation machinery for mito.genes
2. Inner membrane: cristae increases surface area, ETC, ATP synthase, transport proteins, H+ electrochemical gradient (drives ATP synthesis)
3. Outer membrane: porin forms channels
4. Intermembrane space: between inner/out membranes, cytochrome c

5

Role of Mitochondria in Apoptosis

Cytochrome c released from intermembrane space into cytosol --> caspase cascade (proteolytic cascade, cleaves key cellular proteins)

6

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

7

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

8

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

9

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

10

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

11

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)

12

Disorders of peroxisome biogenesis

- defects in proteins required for biogenesis
- lack many peroxisomal enzymes or peroxisomes are absent from cells

13

Zellweger Syndrome

- disorder of peroxisome biogenesis
- peroxisomal enzymes synthesized normally but not imported
- empty peroxisomes
- lethal in early infancy

14

Deficiency of single peroxisomal enzyme

- defect in synthesis, import or function of one peroxisomal protein
- less severe phenotype
- partially functional peroxisomes

15

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

16

Treatment of ALD

1. Allogeneic stem cell transplant: high morbidity, compatible donor cells not always available, must be performed at early stage of brain lesions
2. Gene therapy: recent success in two patients

17

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*