1. Intracellular Compartments and Protein Sorting
September 3, 2019
Tom Gallagher
CTRE, Room 234
extension 64850

2. Intracellular Compartments
See also “Visual Guide to Human Cells”

3. A map of protein trafficking in a eukaryotic cell

4. Sorting signals direct proteins to specific compartments

5. Some Typical Signal Peptides
Function of Signal Peptide
Example of Signal Peptide
Import into ER
Retain in lumen of ER
Import into mitochondrial matrix
Import into nucleus
Export from nucleus
Import into peroxisomal matrix
Usually at the N-terminus; about residues in length, central region is often hydrophobic
Usually at the C-terminus, short 4-residue KDEL is a common ER retention signal
Usually at the N-terminus, about 30 residues in length, often an amphipathic alpha helix
Usually NOT at protein termini; a positive charged sequence is common, i.e., KKKRK
Diverse, but always distinctly different than nuclear import signal, i.e., HLLALKLAGLDI
Usually at the C-terminus, i.e., SKL-COOH

6. Gated Transport between Cytosol and Nucleus

7. The nucleus is bounded by a double membrane that is perforated with nuclear pores

8. The nuclear pore is type of molecular sieve

9. Ran proteins control nuclear import and export

10. Ran proteins control nuclear import and export

11. Nuclear import and export: IMPORTANCE
In cancers: Several human cancers overexpress export receptors; dysregulating import/export
In biological therapeutics: Several experimental therapeutics; i.e., CRISPR/CAS; operate in nuclei (need to know about nuclear import and export)
In cell signaling: Several signaling systems rely on nuclear import for temporal and spatial control

12. Cell signaling : Control at nuclear import stage

14. Transmembrane Transport between Cytosol and Mitochondria

15. The mitochondrion is bounded by two membranes, creating four compartments

16. The mitochondrial matrix import signal peptide and its interaction with the mitochondrial import receptor

17. Protein import into the mitochondrial matrix requires the TOM and TIM complexes

18. Protein import into mitochondria requires chaperones and an electrochemical gradient

19. Mitochondria : IMPORTANCE
Mitochondrial disease: Collection of very rare genetic diseases causing poor growth, developmental delays, muscle weakness
Mutations in the mitochondrial DNA genome are largely responsible; however it is notable that mutations in either mtDNA, or the nuclear DNA genes encoding mt proteins, can cause similar disease symptoms (a case of phenocopying here)
Impaired import of mitochondrial proteins: One clinical example in Huntington’s Disease

20. Mutant huntingtin protein interferes with mt import:
Starves neurons of essential ATP
Inhibition of mitochondrial protein import
by mutant huntingtin
Hiroko Yano1–4, Sergei V Baranov1, Oxana V Baranova1, Jinho Kim1, Yanchun Pan2, Svitlana Yablonska1,
Diane L Carlisle1, Robert J Ferrante1, Albert H Kim2,3,5 & Robert M Friedlander1,6

22. Transmembrane Transport between Cytosol and Peroxisomes

23. Peroxisomes: Organelles for Oxidation Reactions
Oxidation of very long chain and branched fatty acids
Oxidation of cholesterol to bile acids (in liver)
Oxidation (detoxification) of metabolic intermediates; i.e., ethanol oxidation to acetaldehyde
Decomposition of hydrogen peroxide (catalase-mediated decomposition to water and oxygen)
Synthesis of plasmalogens (membrane lipids found in myelin)
catalase crystals

24. Peroxisome precursor vesicles bud from the ER

25. Membrane protein integrase? membrane protein receptor/transporter
Peroxisomal proteins are imported from the cytosol after their synthesis is completed
PTS1 receptor
PTS2 receptor
Membrane protein integrase?
membrane protein receptor/transporter
Free polyribosomes
Protein translocator

26. Peroxisomes : IMPORTANCE
Zellweger Syndrome: Rare autosomal recessive disease (1 in 50,000) caused by mutations in genes encoding peroxisome biogenesis factors;
Absence of peroxisomes causes leukodystrophies (loss of white matter in CNS, due to fewer plasmalogens
Absence of peroxisomes also causes accumulation of fatty acids, in macrophages, these accumulated lipids impair function, especially in CNS macrophages (this is very similar to a lysosomal storage disorder)

27. The correct answer is (D).
Conversion of ethanol to acetaldehyde takes place in peroxisomes and smooth ER microsomes (via ADH (alcohol dehydrogenase)).
Subsequent conversion of acetaldehyde to acetate takes place in mitochondria (via ALDH2 (alcohol dehydrogenase 2).
Disufiram (antabuse) blocks the second reaction (to nontoxic acetate), causing accumulation of toxic acetaldehyde.

29. Transmembrane Transport between Cytosol and Endoplasmic Reticulum
CO-TRANSLATIONAL TRANSPORT

30. Reconstruction of the rough and smooth endoplasmic reticulum

31. About half of protein synthesis is directed to the ER

32. Synthesis of a lumenal (often secreted) protein

33. Synthesis of a single-pass transmembrane protein

34. Synthesis of a double-pass transmembrane protein

35. Synthesis of a multi-pass transmembrane protein
(7-transmembrane G-protein coupled receptors)

36. A smaller proportion are glycosylated on serine or threonine residues
Proteins in the lumen of the ER are glycosylated on asparagine residues in the sequence -Asn-[X]-[Ser/Thr]-
A smaller proportion are glycosylated on serine or threonine residues

37. Protein Folding and Misfolding in the ER:
Membrane and secreted protein folding is challenging.
Mis- or un-folded proteins can build up in the ER, especially in secretory cells (endocrine and some immune cells). This causes an UNFOLDED PROTEIN RESPONSE.

38. UNFOLDED PROTEIN RESPONSE : The sensors
Indeed there are additional quality control mechanisms in place within the ER. These involve three sensors for misfolded proteins, IRE1, PERK, ATF6. Each work to maintain ER homeostasis, by signalling to the nucleus for increased expression of proteins that can improve ER client protein folding.

39. UNFOLDED PROTEIN RESPONSE: Homeostatic mechanism

40. Chronic unfolded protein response : IMPORTANCE
Diabetes: Constitutive insulin secretion; UPR activation; apoptosis of islet cells; insulin dependent diabetes
Several neurodegenerative diseases (Alzheimers, Parkinsons, Huntingtons, Amyotrophic Lateral Sclerosis (ALS)); UPR activation, proteinopathies.
Specific examples: Alpha anti-trypsin deficiency

42. Protein Folding and Misfolding in the ER:
Proteins failing to fold up properly are retro-translocated back into the cytosol and degraded by proteasomes. The desired proteins, therefore, never get “past” the ER
IMPORTANCE:
Cystic fibrosis
Various lysosomal storage diseases (Gaucher disease)