Meeting 10 & 11

• when macrophage takes up bacteria/prey and takes it to the lysosome
• -material targeted for degredation are targeted to the lysosome
• -endocytosis: can also help cell acquire fat

AP complex: interact with receptor, trigger assembly, and trigger endozytosis

• -important for endocytosis...
• ....

process by which a specific receptor on the cell surface binds tightly to an extracellular macro-molecular ligand that it recognizes; the plasma-membrane region containing the receptor-ligand bound complex then buds inward and pinches off, becoming a transport vesicle

particle that has a shell composed of proteins (apolipoproteins) and a cholesterol-containing phsopholipid monolayer

low-density lipoprotiein (LDL): transports cholesterol via blood; composed of shell which is phospholipid monolayer + apoB-100 protein & inside is made up of cholesterol esters

changes in pH

1) LDL receptors bind to apoB protein in the outer layer of

• Rab proteins; ex. Rab5 is a marker for early endosomes
• Rab4: fast recycling loop
• Rab11: essental for recylcing from late endosome
• Rab7: targets degredation?

-Rab's allow fusion of vesicle to a specific target membrane that has specific receptors for Rab

-Early Sorting Complex Required for Transport; required for the recognition and sorting of ubiquitin-modified cargo proteins into the internal vesicles; ubiquitin tags are recognized by the ESCRT complexes, which bind sequentially and work in concert to pass along the cargo proteins from one complex to the next

-Vps4: an ATPase that dissassembles the ESCRT complexes after the vesicle's been pinched off and returns to them to floating around in the cytoplasm

• -proteins that are usually tagged with ubiquitin & recognized by ESCRT: Hrs, which helps load NON-ubiquitinated proteins into cargo buds
• -the HIV protein GAG can immitate its action when tagged with ubiquitin, thereby promoting the transfer of HIV DNA throughout cells using vesicle transport

carries ubiquitin; helps ubiquitinate cargo protein that will go inside; also, protein that recruits ESCRT, which contains Tsg101 that recognizes ubiquitin on Hrs

catabolic process involving the degradation of a cell's own components through the lysosomal machinery; mechanism by which a starving cell reallocates nutrients from unnecessary to more-essential processes

signal in proteins to be sent to the nucleus; such proteins are are fully formed in the cytosol, & specific locations in the nuclear envelope (nuclear pores) are how they're transported inside

-usually Lys enriched

• -very large
• -40-80x bigger than the ribsome
• -there's radial symmetry; repetition of various units forming the 'crown' (view from cytoplasm)
• -view from inside nucleus is different; looks more like a basket

-structure is polarized; two different sides look very different

• should be naturally found in the cytoplasm; important for breakdown of pyruvate
• -if you attach an NLS to pyruvate kinase (something usually found in cytoplasm) and stain for it it'll be found in the NUCLEUS

• treating cells with this permeabilizes the plasma membrane; just makes the cell leak; many of the cytoplasmic components are gone
• -if you add lysate and look at nuclear protein you can see it (if you don't add it, nuclear

-tells us: there are components in the CYTOSOL required for nuclear transport

• type of protein that moves other proteins into the nucleus by binding to the NLS (nuclear localization signal); has 2 subunits, α & β
• -β can bind & transport cargo by themselves or can form heterodimers w/ α
• -in the heterodimer, β interactis with the pore complex & α acts as anadaptor protein to bind NLS on the cargo

GEF is on the nucleic side of things

GAP (GTPase-Activating Proteins): regulatory proteins who bind to activated G-proteins and stimulate GTPase activity = results in a stop of a signaling event

GEF (Guanine nucleotide exchange factor): activate GTPase by stimulating release of GDP to allow binding of GTP

• -proteins are fully synthesized before/WHEN they're
• -mitochondria/chloroplasts are not synthesized de novo: they replicate
• -this has reprecussions for protein targeting; proteins targeted to either of these organelles are targeted to fully formed organelles

contains only a few genes; is a small chromosome that encodes a couple of proteins & machinery mitochondrion needs for transLATION (less so for transcription)

-examples include ribosomal RNA, small tRNAs, couple proteins (Cytochrome oxidase 1 & 2: belong to the last complex & reoxidize cytochrome + reduces oxygen)

proteins that are taken up by mitochondrion will be protected from trypsin degeneration if inside mito; simply left in make-shift cytoplasm, they'll be degraded

• -no co-translational translocation
• -mitochondria has to be energized from (1) ATP or (2) proton-motive force

• •Not identical but they share common motifs
• •need a matrix-targeting sequences
• •found at N-terminus of protein
• •are 20–50 amino acids in length (short)
• •contain hydrophobic aas, basic a.a.s, hydroxylated a.a.s; tend to lack acidic amino acids
• •usually take the shape of an amphipathic α-helix

• 1) protein must have already been translated (are moved POST- translationally)
• 2) proteins MUST be unfolded to enter the matrix (this is done by chaperones [Hsc70] in the cytosol); uses ATP b/c folding is spontaneous, so must counteract
• 3) outer mt membrane has receptor: Tom 20/22 (translocon outer membrane) is a receptor; recognizes signal sequence
• -attracts Tom40, a general import pore; has a beta-strand barrel structure (this is indicative of mt's bacterial origin)

-unsure where energy to PUSH protein through translocon comes from...(b/c already translated)

• 4) Tim (translocon on INNER mt membrane); comes in close contact with Tom ['contact site']; protein goes through both ~at the same time
• 5) binding of Hsc70 to protein INSIDE matrix helps with import in matrix
• 6) matrix protease cuts uptake-targeting sequence

have signals in primary sequence, directs them to inner membrane via 3 paths

(A) this type of protein has matrix-targeting sequence (cleaved by matrix protease) & hydrophobic STS (stop transfer sequence; which sticks when passing through Tim channel)

(B) this type of protein has matrix-targeting sequence (cleaved by matrix protease) + sequences recognized by Oxa1 (inner-membrane protein)

(C) this type of protein DOESN'T have a matrix-targeting sequence, but instead has multiple internal mt targeting sequences, which are (hydrophobic) and arranged in the inner membrane by Tim complexes

(A) this type of protein has matrix-targeting sequence that's 1st cleaved by matrix protease & then an intermembrane-space-targeting sequence is cleaved 2nd by proteases in intermembrane space

(B) this type of protein only has a targeting sequence for general import port (Tom40)/intermembrane space, so it stays there

this type of protein (ex. Tom40) has the simplest path; comes from the outside & only need to be put at that membrane; first interact with general import pore (Tom 40), then SAM [sorting & assembly machinery] complex; they have a short matrix-targeting signal (?) & a (hydrophobic) stop-transfer/outer-membrane localization sequence

• -NEITHER signal sequence is cleaved
• -mechanism/energy source/how they interact with SAM is unclear

• -is circular; proteins it encodes are made in stroma (inside inner membrane but outside thylakoid), but most are encoded by nucleus
• -an example of protein that has some subunits encoded in chloroplast & some in nucleus = RUBISCO; has 8 large subunits made in chloroplast, & 8 small ones made in nucleus

1) SRP-dependent pathway (plastocyanin): similar to how proteins are targeted to mt, proteins have both a stromal-import & thylakoid-targeting sequence; 1st go through Toc (outer membrane) then Tic (inner membrane); once in stroma import sequence is cleaved, & SRP binds to thylk-targeting sequence to import through SecY translocon...now protein's in lumen

2) pH pathway (metal binding proteins): same as above except once protein's in stroma & import sequence has been cleaved, metal ions are added to unfolded protein, thereby folding them; need (a) 2 argenine residues (RR) & (b) pH gradient for subsequent transport of protein into inner lumen.....argenines are cleaved once in lumen

copper-containing protein involved in electron-transfer (now you know it's found in the lumen of the thylakoid)

• •single membraned
• •have no DNA
• •Compartment of Oxidation; whenever there’s oxidative
• stress/destroy things via oxidation
• •2 O2 + 2 S → 2 H2O2 + 2 S(ox)
• •Contains catalase: 2 H2O2 → 2 H2O + O2
• •has peroxisomal signal sequences
• –PTS1: located at the C-terminus Ser-Lys-Leu (SKL)
• –PTS2: N-terminus ???

-where oxidation occurs, b/c they create hydrogen peroxide

catabolize fatty acid chains, branched chain fatty acids (+ D-amino acids, polyamines, biosynthesis of plasmalogens, etherphospholipids) & necessary for normal brain and lung function

• 1) proteins are fully folded in cytosol; PTS1 is found at the C-terminus (like KDEL, different from practically everything else)
• 2) Pex5, a soluble receptor floating in cytosol recognizes PTS1 (again, different mechanism, NOT a transmembrane receptor)
• 3) Pex5 interacts with a receptor (Pex 14) on peroxisome membrane; subsequently transported in through another membrane protein (Pex10/12/2)
• 4) in the matrix Pex5 dissociates and is transported back to the cytosol via same Pex10/12/2 complex

yay!