1. Overview of the cell structure Eucaryotic cell Organelles

2. Readings and Objectives Reading – Cooper: Chapter 1, 9, 10 (p.408), 11(p.464) Objectives – Basic properties of eucaryotic cells – Eucaryotic organelles Nucleus Ribosomes ER and Golgi Lysosomes and peroxisomes Note: Cell membrane, endosomes, mitochondria, and cytoskeleton are separate lectures

3. Eucaryotic Organelles The Nucleus 5-10 µm diameter: largest organelle; contains the linear DNA molecules nucleus separated from the cytoplasm by the nuclear membrane. nuclear pores : import and export of RNA and proteins Nucleolus: sites of rRNA synthesis and ribosomal subunits maturation

4. Nucleus and Ribosomes Nucleus contains chromatin within a semifluid nucleoplasm. Chromatin: composed of DNA, protein, and some RNA, is usually a network of fine strands. condense during cell division to form visible chromosomes

5. Nuclear Envelope Nuclear envelope separates nuclear content from the cytoplasm selective traffic of proteins and RNAs critical in regulating eukaryotic gene expression Consists: two membranes, underlying nuclear lamina, and nuclear pore complexes outer membrane- continuous with the endoplasmic reticulum, has membrane proteins that bind the cytoskeleton inner membrane has proteins that bind the nuclear lamina Each nuclear membrane is a phospholipid bilayer permeable only to small nonpolar molecules.

6. Nuclear Lamina Nuclear lamina- fibrous mesh that provides structural support. fibrous proteins called lamins Mammalian cells have three lamin genes, (A, B, and C) Two lamins interact to form a dimer in which the α-helical regions of two polypeptide chains wind around each other to form a coiled coil The lamin dimers associate with each other to form the nuclear lamina Lamina

7. Nuclear Pore complex Nuclear pore- sole channels for small polar molecules, ions, proteins, and RNA to pass through the nuclear envelope Nuclear pore complexes- composed of ~30 different pore proteins (nucleoporins). Two mechanisms: – Passive transport- small molecules pass freely in either direction – Active transport- Macromolecules (proteins and RNAs), energy- dependent

8. Nuclear Pore complex pore complex consists of eight spokes connected to rings at the nuclear and cytoplasmic surfaces. The spoke-ring assembly surrounds a central channel. Protein filaments extend from the rings, forming a basketlike structure on the nuclear side. Proteins that must enter the nucleus have amino acid sequences called nuclear localization signals. These are recognized by nuclear transport receptors (importins)

9. Nuclear importation importin binds to the NLS of cargo protein complex binds to the cytoplasmic filaments of the pore complex Transport proceeds through pore complex by binding to nucleoporins cargo/importin complex is disrupted by binding of Ran/GTP. conformation change in the importin, releases the cargo into the nucleus

10. Nuclear importation importin-Ran/GTP complex exported back to the cytoplasm where the GTP is hydrolyzed to GDP The importin is released and can participate in another round of transport. Ran/GDP is transported back to the nucleus by its own import receptor (NTF2), where Ran/GTP is regenerated.

11. Ran/GTP Gradient Activity of importins regulated by Ran, a GTP- binding protein. Ran GAP: a GTP hydrolysis to GDP are on the cytoplasmic side of the nuclear envelope enzymes for exchange of GDP for GTP are on the nuclear side. This leads to higher concentration of Ran/GTP in the nucleus, and determines the directionality of transport.

12. Nuclear Export Proteins are targeted for export from the nucleus by specific amino acid sequences, called nuclear export signals. These signals are recognized by receptors in the nucleus (exportins), which direct protein transport to the cytoplasm. Ran is required for nuclear export as well as import. Ran/GTP promotes binding of exportins and their cargo proteins, but dissociates complexes between importins and their cargos.

13. Protein Import and Export through the Nuclear Pore Complex Protein Import and Export through the Nuclear Pore Complex

14. Ribosome Ribosome subunits, one large and one small, are assembled in the cytoplasm and used to make proteins found in both prokaryotes and eukaryotes – A nucleoprotein complex – Assembled in two subunits – Eukaryotic: 80S (60S+40S subunits) – Prokaryotic: 70S (50S+30S) Ribosomes Bacterial 70S ribosome 16S rRNA 5S rRNA 23S rRNA tRNA

15. Components of Eucaryotic and Procaryotic Ribosomes Want to read more? In Slide show Click to go to the book sectionClick to go to the book section 80S; 4 RNA + 83 proteins (in 2 subunits) 70S; 4 RNA + 55 proteins (in 2 subunits)

16. In eukaryotic cells, ribosomes can be found in different locations and forms. Single free-ribosomes in the cytoplasm – Grouped into polyribosomes, make cytosolic proteins Attached to the endoplasmic reticulum, rough ER (RER), make proteins targeted to membranes and organelles mRNA, produced in the nucleus Transported to cytoplasm by carrier proteins, pass through nuclear pore Ribosomes

17. Ribosomes, ER and protein synthesis Ribosomes are the protein synthesis machinary

18. Ribosomes, ER and protein synthesis Proteins translated on RER Post-translationally modified in golgi eg. glycosylation Targetted to other organelles Cell membrane Vesicular compartments

19. Endomembrane system: ER and Golgi Endomembrane system ER: tubular membranes and cisternae Smooth ER-lipid and steroid synthesis RoughER- protein synthesis Together with Golgi form the cellular secretory machinary

20. Flattened membranous sacs Chemically modifies proteins from rough ER, Sorts finished proteins to their destiny site of lipid synthesis, and (in plant cells) the site of synthesis of some polysaccharides that compose the cell wall Golgi Complex cis face associated with ER movement of materials

21. Flattened membranous sacs Chemically modifies proteins from rough ER, Sorts finished proteins to their destiny site of lipid synthesis, and (in plant cells) the site of synthesis of some polysaccharides that compose the cell wall Golgi Complex

22. Lysosomes Golgi derived membrane-bound vesicles (0.1-1.5 um) found in most eucaryotes involved in intracellular digestion and recycling of macromolecules contain about 40 acid hydrolases – proteases, nucleases, and phopholipases, amaylases maintain an acidic environment by pumping protons into their interior (pH,5.0) Digest worn out cellular molecules, engulfed bacteria and viruses – Fuse with phagosomes (eg engulfed bacteria) to form phagolysosomes Lysosomes also participate in apoptosis, or programmed cell death Tay-Sachs disease: lysosomal disfunction, autosomal recessive – Defective Hexosamidase A, a hydrolase involved in breakdown of phospholipids – Accumulation of lipids in neurons, infantile death Vesicular compartments

23. Peroxisomes single-membrane-enclosed microbodies Originate form ER, cytosolic proteins are imported to peroxisomes (no golgi origin) contain enzymes involved in variety of oxidative reactions peroxisomal proteins (peroxins) are metabolic enzymes lipids broken down by oxidative reactions  H2O2 Peroxisomes also contain catalase Involved in synthesis of amino acids Synthesis of cholesterol, bile acids Detoxification rxn, eg alcohol Vesicular compartments

24. Peroxisomes: assembly Peroxisome assembly begins on the rough ER, where two peroxins, Pex3 and Pex19, initially localize. Their interaction causes Pex3/Pex19-containing vesicles to bud off the ER. The vesicles may then fuse with preexisting peroxisomes or with one another to form new peroxisomes.

25. Peroxisomes peroxins synthesized on free ribosomes and imported Protein import and the addition of lipids from the rough ER results in peroxisome growth, and division Enzyme content and metabolic activities of peroxisomes changes as they mature Zellweger syndrome, named after Hans Zellweger (1964) Pex1,2,3,5,6,12,14 and 26 mutated (1:50,000) Inefficient peroxisomal protein import Long fatty acid chains accumulate in liver and neurons Neurological disorders, glaucoma, hepatic enlargement, mental incapacity, seizure, loss of muscular tone, facial deformities and Jaundice lethal within a few years See website literature section for a review article

26. Proteosomes and protein degradation after release some vesicles deliver their contents to lysosomes while others deliver to cell membrane Quality assurance mechanism – Unfolded or misfolded proteins are secreted into cytosol, targeted for destruction by ubiquitin polypeptides – proteasomes destroy targeted proteins

27. Proteosomes and protein degradation