lecture stuff

nuclear envelope separates the nucleus from the cytoplasm, but it’s not completely closed — molecules must enter and leave through nuclear pores

Nuclear pore complexes allow selective transport. Only small molecules (<5000 Da) can diffuse freely. Larger molecules (like proteins or RNA) need active transport. The membrane protects DNA from “leaking” into the cytoplasm. So, nuclear pores are gated channels that control molecular traffic.

Nuclear Localization Signal (NLS) → directs import of proteins into the nucleus (e.g., transcription factors). Nuclear Export Signal (NES) → directs export (e.g., mRNA, rRNA, tRNA, ribosomal subunits). These are recognized by transport proteins called importins and exportins.

experiment showing exchange of nuclear materials between amoebae. Labeled one amoeba’s nucleus radioactively, transplanted it into another. Over time, labeled nuclear molecules moved into the host nucleus. This showed that some nuclear proteins shuttle back and forth — implying separate NLS and NES signals exist.

the first protein used to study nuclear import. A pentameric (five-part) nuclear protein found in frog eggs. Helps assemble nucleosomes (DNA + histones). Its import into the nucleus was found to depend on a specific sequence — the NLS.

They purified nucleoplasmin and labeled it radioactively. Cut it into “heads” and “tails” to test which part carried the NLS. Found that only the tails entered the nucleus → the NLS is in the tail region. (reinforces the idea that a short amino acid sequence can direct nuclear import.)

tested if the NLS alone is sufficient to direct import: Attached nucleoplasmin tails to gold particles (electron-dense for TEM imaging). The gold particles entered the nucleus → proving that the NLS is sufficient to direct nuclear import, even for something non-protein like gold. However, we still need import machinery to recognize and move NLS-tagged cargo.

Shows the Ran/importin system: Importin binds the NLS on a protein → carries it through the nuclear pore. Inside nucleus, Ran-GTP binds importin → cargo released. Importin–Ran-GTP exits the nucleus. In the cytoplasm, Ran-GTP → Ran-GDP (by GAP enzyme), releasing importin. Ran-GDP is re-imported and converted back to Ran-GTP (by GEF enzyme). This Ran cycle powers directional transport — ensuring cargo moves into the nucleus, not randomly.

Short and largely unsaturated acyl chains that tend to kink and pack more loosely allowing rapid lateral movement (fluidity

Long and largely saturated acyl chains allows tight packing (gel- like)

Cholesterol interacts preferentially (but not exclusively) with sphingolipids, and favours phase separation between sphingolipids and glycerophospholids

Predominantly sphingolipid and cholesterol-rich microdomains in the outer (exoplasmic) leaflet of the membrane (…composition can change…) • Cholesterol and glycerophospholipids also present in inner leaflet • GPI-anchored, acylated & transmembrane proteins also present in raft Raft “lifetime” can vary from ns to mins (i.e. signaling) Lipid raft platforms can range from 10-1000 nm

• Signal transduction • Lipid rafts for adhesion and changes in shape during migration • Help form the blood-brain barrier Disease: * Cancer cells have a higher level of. lipid rafts * Amyloid plaque accumulation -Alzheimer’s * Lipid rafts can be targets of different pathogens * Raft localization of cellular prion protein which facilitates conversion to pathological form

Dependent on the ability of leukocytes to: * migrate to regions of infection (inflamed tissues) * interact with other cells during antigen presentation * respond to the inflammation

Because some proteins need specific environments to function

A signal or tag that says “send me to the peroxisome For peroxisomes, this signal is called a peroxisomal targeting signal (PTS)

the biotech company should add a peroxisomal targeting signal (PTS) to the enzyme’s amino acid sequence The cytosol doesn’t provide the right environment for the enzyme’s function — for example, peroxisomes contain oxidative enzymes and detoxifying conditions that are essential for breaking down fatty acids or reactive oxygen species. If the enzyme stayed in the cytosol, it might misfold, be inactive, or harm the cell. You can engineer the enzyme to include a peroxisomal targeting sequence (PTS): PTS1: A short C-terminal sequence (typically Ser-Lys-Leu, or SKL) These sequences are recognized by peroxins (PEX proteins) such as PEX5, which bind the signal and import the protein into the peroxisome. Tag the enzyme with GFP (green fluorescent protein) and visualize yeast cells under a fluorescence microscope. If the enzyme localizes to small, punctate peroxisomes, it worked. If the fluorescence fills the cytosol, it didn’t. Alternatively, perform biochemical fractionation to isolate peroxisomes and check for the enzyme’s presence using western blotting.