What is the “diffusion problem” and how is it solved in eukaryotic cells?

• Problem: As the cell increases in diameter, its volume increases more than its surface area therefore the rate of exchange is decreased, diffusion only allows for rapid movement across short distances
• Solved: because the eukaryotic cells are highly compartmentalized the cytosol is only a fraction of total cell volume reducing effect of total cell surface-to-volume ratio

What components are found only in eukaryotic cells? What are the functions of these components?

• Mitochondria - produces ATP
• Nucleus - holds DNA
• Golgi Apparatus - synthesizes RNA
• lysosomes- recycling bin
• membrane bound organelles
• Microtubules
• ER - rough + smooth
• peroxisome- centers for reduction oxidation reactions

How does the nuclear envelope differ from the plasma membrane? How do molecules move between the cytoplasm and the nucleus?

• Nuclear envelope: is supported by fibrous proteins that form a lattice-like sheet called the nuclear lamina which stiffens and shapes the structure
• Plasma membrane:
• Through the nuclear pore complex mediates the selective exchange of components including RNA, ribosomal proteins, signaling molecules, and lipids between the nucleus and cytoplasm
• Through nuclear pores, nuclear localization signal (NLS)

Describe the experiment that suggested that a particular sequence of amino acids served as a kind of “zip code” to direct large proteins, such as nucleoplasmin, into the nucleus? Is the uptake of nucleoplasmin tails into the nucleus active or passive transport? How can you tell?

• Labeled nucleoplasmin (strictly found in nucleus) with a radioactive atom then injected it into the cytoplasm of the cells. It quickly then went to the nucleus.
• Then researched used enzymes to separate the core section and tails, labeled each w/ radioactive atom, then injected them into cytoplasm of diff cells
• The tail ends entered the nucleus quickly but the core could not enter
• hypothesis: Nucleoplasmin contains a discrete “send to nucleus “ signal that resides in either the tale or core region
• null hypothesis: nucleoplasmin doesn’t require a signal to enter the nucleus, or the entire protein serves as a signal
• prediction: labeled tail region or labeled core region will be found in the nucleus
• Null hypothesis prediction: Either both (no protein signal) or neither (whole protein signal)
• conclusion: send to nucleus signal is in the tail region of the nucleoplasmin
• Active because it goes against its concentration gradient.

Are signal sequences unique to nuclear proteins? If not, where else can proteins be targeted? Are the signal sequences on proteins targeted to these different compartments the same?

No, other proteins have signal NLS extracellular proteins

• Phosphorylated sugars serves as a zip codes for lysosome bound proteins

What are the main components of the endomembrane system?

• Golgi Apparatus - cis surface (closest to nucleus) receives products from rough ER. Trans side (oriented towards plasma membrane) ships products out to other organelles or cell surface + beyond. in b/t within cisternae products are processed + packaged for delivery. Ex: Acid hydrolysis processed in Golgi them shipped to lysosome
• Lysosomes - Recycling centers that hydrolyze different macromolecules and send to cytosol for new energy
• Endoplasmic reticulum - Acid hydrolysis are synthesized here processed in the golgi and then shipped to the
• Producing, processing, and transporting proteins and lipids in eukaryotic cells

What is the secretory pathway hypothesis? How was the secretory pathway hypothesis experimentally confirmed?

Protein enters RER, protein exits RER in a vesicle, protein enters golgi apparatus, protein exits golgi apparatus, protein is secreted from cell in a vesicle

What is meant by a “pulse-chase” experiment?

• The Pulse-Chase Method: originated from observation by George Palade of large amounts of Golgi apparatus and ER found in cells that secrete digestive enzymes, hormones, or etc. by looking through electron micrographs. Lead to hypothesis stating that cells partake in a “secretory pathway” that starts in the ER and ends in the extracellular membrane.
• Pulse - expose cell to high concentration of amino acids for a short period of time (Ex. radiolabeled amino acid)
• Chase - time following the end of the pulse. the pulse ends by washing away the modified amino acid and replacing it with the normal version of the same molecule. If the chase consists of unlabeled amino acid, then the proteins synthesized will not be radiolabeled.
• Intention - mark a particular group during an interval and follow their changes (if any) overtime specifically with pancreatic cells growing in culture which are usually packed with ER and golgi and are used in small intestine to secrete digestive enzymes
• Results - Showed that proteins are trafficked through the secretory pathway from RER->Glolgi ->extracellular in about two hours

Where in the cell are proteins synthesized? What is the “signal hypothesis,” and how was the signal hypothesis experimentally confirmed for proteins that enter the lumen of the ER? How are proteins that are produced in the ER transported to the Golgi?

Proteins are synthesized in ribosomes free in the cytosol -> eventually these ribosomes become attached to the rough ER

Signal Hypothesis

• First few amino acids in the growing polypeptide chain act as a signal that marks the ribosome for transport to the ER membrane

• Realized that when proteins that are normally synthesized in the rough ER are manufactured by free floating ribosomes instead they are on average 20 more amino acids longer

• Prediction: Proteins bound for the endomembrane system have a molecular zipcode analogous to nuclear localization signal (NLS)
• Results: Extra amino acids on free floating ribosomes are the “send to ER” signal called ER signal sequence - will move proteins to ER lumen

Proteins are transported from the ER to the Golgi by vesicles that bud off from the rough ER and carried to the golgi apparatus with only the appropriate cargo and then fuse with the membrane of the cis face of the golgi (side nearest rough ER)

What is “receptor-mediated endocytosis?” Draw the pathway that moves molecules from the extracellular space to the lysosome.

Receptor-mediated endocytosis is the most specific type of endocytosis. The main difference between receptor-mediated endocytosis and phagocytosis is that rather than the cell membrane’s specific protein receptors attaching to the receptors of the bacterial cell like in phagocytosis, in receptor-mediated endocytosis the actual molecule that is being ingested binds directly to the receptors of the cell membrane (ex. macromolecules like sugars and hormones) pinching off to form a vesicle and fuses with an early endosome to lower it’s pH and release it’s cargo before being digested and recycling its membrane proteins for another cycle of receptor-mediated endocytosis.