Overview of the cell structureOverview of the cell structureEucaryotic cell OrganellesEucaryotic cell Organelles

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

Eucaryotic OrganellesThe NucleusThe 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

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

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.

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

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

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)

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

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.

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.

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.

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

• 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)

RibosomesRibosomes

Bacterial 70S ribosome

16S rRNA 5S rRNA

23S rRNA

tRNA

Components of Eucaryotic and Procaryotic RibosomesComponents of Eucaryotic and Procaryotic Ribosomes

Want to read more? In Slide show Click to go to the book section

80S; 4 RNA + 83 proteins (in 2 subunits)

70S; 4 RNA + 55 proteins (in 2 subunits)

• 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

RibosomesRibosomes

Ribosomes, ER and protein synthesis Ribosomes, ER and protein synthesis

Ribosomes are the protein synthesis machinary

Ribosomes, ER and protein synthesis Ribosomes, ER and protein synthesis

• Proteins translated on RER

• Post-translationally modified in golgi eg. glycosylation

• Targetted to other organelles• Cell membrane• Vesicular

compartments

Endomembrane system: ER and GolgiEndomembrane system: ER and GolgiEndomembrane systemEndomembrane system• ER: tubular membranes and

cisternae• Smooth ER-lipid and steroid

synthesis• RoughER- protein synthesis

• Together with Golgi form the cellular secretory machinary

• 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

• 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

LysosomesLysosomes• 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

PeroxisomesPeroxisomes• 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

Peroxisomes: assemblyPeroxisomes: 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.

PeroxisomesPeroxisomes• 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 yearsSee website literature section for a review article

Proteosomes and protein degradationProteosomes 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

Proteosomes and protein degradationProteosomes and protein degradation