Notes on abbreviations: most abbreviations are mentioned by their full name at least once prior. Any weird abbreviations I used are noted also on the end of each post.
- organelles are characterized/identified by the proteins on their membrane and in their lumen
- enclosed by intracellular membranes to separate and specialize different reactions/pathways
- also need to be able to export products and import necessary proteins
- nucleus holds DNA, and functions in DNA/RNA synthesis
- cytoplasm is cytosol plus all the organelles
- cytosol is the aqueous volume of a cell, site of protein synthesis and degradation
- ER make soluble and integral membrane proteins and lipids
- Golgi receives proteins and lipids from ER, modifies them, and ships to correct destination
- mitochondria/chloroplasts generate energy as ATP
- lysosomes digest macromolecules and organelles
- endosomes transport endocytosed stuff to lysosomes
- peroxisomes sequester dangerous oxidation reactions
- organelles also characterized by position within cell (e.g. Golgi close to nucleus)
- organized by cytoskeleton interactions
- precursor of eukaryotic cells had no internal membranes and performed all membrane functions on the plasma membrane
- adaptation to internal membranes increased surface area available for all these reactions
- adaptation to specialization of internal membranes segregated reactions for more efficiency
- adaptation to enlargement of eukaryotes (much bigger than lil' bacteria) in conjunction with efficiency of energy, storage, transport, etc.
- most likely evolved from simple folding in (invagination) of membranes and subsequent pinching off into vesicles--this process still occurs in terms of transporting goods from one organelle to the next
- mitochondria and plastids evolved from endosymbiosis (evidenced by their own separate DNA)
- sorting signal: part of A.A. sequence of polypeptide of protein, directs delivery of newly synthesized protein to location out of cytosol (into an organelle for example)
- proteins without sorting signals remain in cytosol permanently
- 3 methods of protein transport:
- gated transport: protein channel that only allows passage of transporters, molecules need transporters to move them through channel (ex. NPC of nucleus)
- used for transport between spaces that are similar (nucleoplasm and cytoplasm)
- transmembrane transport: protein channel that allows passage of molecules with correct signal, molecules that are supposed to go through unfold to go through (ex. movement into ER or mito)
- used for transport between spaces that are not similar (ER lumen and cytoplasm)
- vesicular transport: comparatively less selective, membrane buds off and takes whatever is in its lumen into another compartment, inside goods never see what's outside the membrane
- used for transport between spaces that are similar but requires going through space that is not similar (from ER lumen to Golgi lumen but through cytoplasm)
- signal sequence: A.A. sequence at the N-terminus that contains the sorting signal
- signal peptidase cleaves the sequence once the protein has been delivered to the destination
- signal patch: a type of permament signal sequence in the middle of a polypeptide that forms a 3D area when the protein is folded and is not cleaved (e.g. NLS or NES for nuclear transport)
- ER signal is usually 5-10 hydrophobic residues at the N-terminus
- not continuing to Golgi involves a specific sequence of 4 residues at the C-terminus
- mitochondria signal is alternating positive and hydrophobic residues
- sequences are identified by using site-directed mutagenesis and see where proteins end up after you mutate a part of its sequence (if you mutate a section of the sorting signal, the protein goes somewhere else, and then you sequence that important part to identify the sorting signal)
- same destinations may have different sequences: characteristics like hydrophobicity are more important than the actual A.A.
- similarly, receptors recognize classes of sequence signals rather than be specific
- 3 methods of studying protein translocation:
- transfection: fuse cytosolic protein with a signal sequence, transfect cell with the cDNA of this fusion protein, let the cell express it, determine where the protein ends up by immunostaining or cell fractionation. THEN, do site-directed mutagenesis to see which residues of the sequence are most important.
- biochemistry: perform in vitro translation of a protein that has the signal of interest (SgOI), label with radioactive A.A., place in proximity of isolated organelle, and see if it translocates
- it translocates into the organelle if:
- 1. labeled protein cofractionates with organelle in centrifugation
- 2. isolated protein moves faster than control protein through gel because the organelle cleaved the signal
- 3. can still isolate intact protein when you add nucleases because the organelle is protecting it, but detergents that remove the membrane allows nuclease digestion)
- genetics: engineer mutations in the translocation machinery, wait to see if cell dies because an important protein went somewhere else or couldn't go to destination at all
- most organelles cannot be made de novo
- some can be produced from budding off of others
- neither can specific translocation machinery be produced from scratch
pg. 695-704 (from Chapter 12)
A.A = amino acid
