1. Intracellular Compartments and Protein Sorting
Transport of Proteins into Mitochondria
and Chloroplasts
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3. Subcompartments of Mitochondria and Chloroplasts
Both organelles contain their own DNA and ribosomes
Each imported protein must reach the correct subcompartment

4. Mitochondrial Signal Sequence
-Mitochondrial Proteins are first fully synthesized in the cytosol and then translocated into the mitochondria
-The signal sequence is located at the N terminus, which is not a specific amino acid sequence.
-The signal is recognized by receptor proteins on the mitochondria

5. Protein and Receptor
-Alcohol dehydrogenase and mitochondrial import receptor

6. Mitochondrial Protein Translocators
Translocase of the Outer Membrane

7. Mitochondrial Protein Synthesis
1. Mitochondrial proteins do not fold into their native structures following translation in the cytosol
2. They remain unfolded through interactions with other proteins including hsp70 chaperone proteins and others
3. The interacting proteins prevent the proteins from aggregating or folding up spontaneously before they engage with the TOM complex

8. Proteins Spanning Mitochondrial Membranes
This demonstrates that the precursor proteins can pass through both mitochondrial membranes at once to enter the matrix

9. Protein Import Contact Site
Coupling of TOM and TIM complexes at contact sites

10. Role of Energy in Protein Import
Mitochondrial protein import is fueled by ATP hydrolysis

11. Integration of Porins
-B-barrel (pore-forming) proteins that are freely permeable to ions and metabolites, but not proteins
-SAM complex inserts the proteins into the outer mitochondrial membrane
-This pathway is conserved in bacteria

12. Protein Import Driven by hsp70, Thermal Ratchet Model

13. Protein Import Driven by hsp70, Cross-Bridge Ratchet Model
In both models, hsp70 functions as a ratchet that prevents backsliding of the polypeptide chain

14. Protein Import into the Inner Mitochondrial Membrane

15. Protein Import into the Mitochondrial Intermembrane Space and Inner Membrane
Metabolite-specific carrier proteins,
Transport of ADP, ATP, or phosphate

16. Transport into the Thylakoid of Cholorplasts

17. Translocation into the Thylakoid Space or Thylakoid Membrane
Hydrophobic
sequence

19. Characteristics of Peroxisomes
-They are surrounded by a single membrane
-They do not contain DNA or ribosomes, so all proteins must be imported
-They are found in all eucaryotic cells
-They contain oxidative enzymes, sometimes at very high concentrations that they appear crystalline in structure
-In liver and kidney cells, they detoxify various toxic molecules that enter the bloodstream
-They perform the breakdown of fatty acids called
Beta-oxidation

20. EM of Peroxisomes
Paracystalline inclusions

21. Plasmalogen Structure
-Animal peroxisomes catalyze the first reactions in plasmalogen formation
-Are abundant in myelin sheaths of axons

22. Peroxisomes in Plant Cells
Tobacco leaf
Fat-storing cell of a tomato seed
Glyoxysomes are peroxisomes which specifically convert fats (B-oxidation) to sugars (gluconeogenesis)

23. Signaling in Peroxisomes
-The targeting signal is –Ser-Lys-Leu-COO-
-Soluble receptor proteins in the cytosol that recognize the signal sequence and docking proteins on the cytosolic surface of the peroxisome are involved
-Transport is driven by ATP
-Proteins do not need to be unfolded to be imported and may be similar to nuclear import
-Proteins that are involved in the peroxisome signaling process are called peroxins
-Zellweger syndrome – a defect in importing proteins into peroxisomes. Patients have abnormal brain, liver, and kidneys, and die soon after birth

24. Model for Production of New Peroxisomes

25. TABLE 12–3 Some Typical Signal Sequences
FUNCTION OF SIGNAL SEQUENCE EXAMPLE OF SIGNAL SEQUENCE
Import into nucleus -Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-
Export from nucleus -Leu-Ala-Leu-Lys-Leu-Ala-Gly-Leu-Asp-Ile-
Import into mitochondria +H3N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe- Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu- Leu-
Import into plastid +H3N-Met-Val-Ala-Met-Ala-Met-Ala-Ser-Leu-Gln-Ser- Ser-Met-Ser-Ser-Leu-Ser-Leu-Ser-Ser-Asn-Ser-Phe-Leu- Gly-Gln-Pro-Leu-Ser-Pro-Ile-Thr-Leu-Ser-Pro-Phe-Leu- Gln-Gly-
Import into peroxisomes -Ser-Lys-Leu-COO–
Import into ER +H3N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly- Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys- Cys-Glu-Val-Phe-Gln-
Return to ER -Lys-Asp-Glu-Leu-COO–

27. 3 Main Functions of the ER
1. Site of protein synthesis
-Transmembrane proteins
-Water-soluble proteins (cell exterior)
2. Site of lipid biosynthesis for most of the organelles
3. Storage of calcium in the ER lumen

28. EM of Rough ER
Pancreatic exocrine cell

29. Smooth ER
-a point where vesicles bud from the ER in transport to the Golgi
-Prominent in cells which specialize in lipid metabolism, synthesize steroid hormones, and in cells involved in detoxification reactions

30. Isolation of Rough and Smooth Microsomes from the ER

31. Purified Rough ER Fraction

32. Co- and Post-Translational Protein Translocation

33. Normal Levels of Translation
A 10AT 578T 721
M C N C N C N C N C N Rb
110 kD
Pol delta kD
Pol eta
79 kD
Lamin A/C
67, 75 kD
Pol beta
40 kD
PNCA
30 kD

34. Polymerase Compartmentalization
A AT T
M C N C N C N C N C N
Pol δ
Lamin A/C