Part B: Lecture 1 + 2 + 3 + 4 + 5 Flashcards
two ways compartmentalization was achieved
-encased in membrane
-independent of membranes = use molecular environment to create components
why is cellular compartmentalization important?
-increases likelihood of reactions occurring by increasing conc. of rxns in given environments
-prevents side rxns & contamination increasing efficiency
2 factors drive chemical rxns
-conc. of reactions
-the environment
molecules that have _______ characteristics form membranes
hydrophobic & hydrophilic
Stage A of evolution of cell compartmentalization (development of PM)
1) primordial cells may have been vesicles formed by lipid interactions
2) monotopic membrane proteins integrate into those vesicles via insertion sequences
3) vesicle act like capturing device for proteins involved in DNA replication, RNA transcription, protein translation, ribosome assembly
4) Alternations in membrane/ribosome interactions allowed for the integration of bitopic integral membrane proteins
Stage B + C of evolution of cell compartmentalization (Further compartmentalization into closed system)
1) while bitopic proteins are imbedded in membrane, cotranslational translocation develops -> colonization of opposite leaflet by monotopic proteins
2) development of transport system across membrane; cell can communicate with outside world
3) concentration of reaction machinery and prevents interfering rxns
Stage D of evolution of cell compartmentalization (Forming intracellular compartment)
creation of endoplasm compartment and periplasmic space similar to the inside of the precellular vesicle (like Gram - bacteria)- two membrane
Stage E of evolution of cell compartmentalization (Loss of second membrane)
loss of outer membrane could lead to gram + bacteria and precursor for euk cell
Eukaryotic cell compartmentalization is controlled largely _____
by membranes
The membrane of euk cells could be traced to two distinct sources: ______ (2)
1) Invagination of PM of primordial cells + fusion
2) Symbiotic capture of another cell
Invagination of PM of primordial cells for further eukaryotic cell compartmentalization steps
1) nonrandom removal of distinct membrane functions & forming membranes with ectoplasmic compartments
2) formation of nuclear envelope & ER which divides endoplasm into cytoplasm and nuceloplasm, creating double membrane nucleus
3) development of membrane systems like Golgi, secretory vesicles, endocytic vesicles, lysozomes
Symbiotic capture of another cell for further eukaryotic cell compartmentalization steps
1) capture and fusion leads ti double membrane organelles (ex. mitochondria and chloroplasts)
2) Outer mitochondrial and chloroplasts membranes are from the host
3) Inner mitochondrial and inner chloroplasts membranes (+ thylakoid membrane) are from the foreign cel
Protein translocation (def.)
a process by which proteins move between cellular compartments
Types of translocation + def.
cotranslational: coupled to translation.
posttranslational: translocation occurs following synthesis
general structure of mitochondria
-double membrane organelles
-inner membrane
-cristae
-matrix
-outer membrane
outer mitochondrial membrane descr. + function
- membrane contains porin, a protein that forms large channels
- allows the passage of molecules up to 5000 daltons to cross the membrane
- also has lipid synthesis and other enzymes
intermembrane mitochondrial space descr. + function
-beaneath outer membrane
-porin channels allow movement of small molecules across outer membrane so the small molecule composition of space is similar to cytoplasm
inner mitochondrial membrane descr. + function
-inner membrane forms extensive cristae (flattened adjacent membrane) which increase SA of inner membrane
-site of oxidative reactions involved in respiratory chain; location of ATP synthase
mitochondrial matrix descr. + function
-contains enzymes involved in oxidation of pyruvate and the citric acid cycle
Oxidative metabolism in cells is performed by ____, which serves as the major ______ in eukaryote cells. This is accomplished ultimately by the oxidation of _____ in the citric acid cycle leading to the production of _____. The reduction of NAD to NADH provides a source of high energy electrons used in a ______. The proton gradient essentially represents a form of stored energy that can be used by ________. ATP is then transported to the cytoplasm.
-mitochondria
-energy-converting organelle
-Acetyl – CoA
-NADH & FADH2
-chemiosmotic coupling reaction
-ATP synthase to convert ADP → ATP
The mitochondrial matrix also contains the mitochondrial _____
DNA genome, mitochondria ribosome, tRNA, etc
Mitochondrial proteins are targeted to several specific locations (4)
1) Outer membrane
2) Intermembrane space
3) Inner membrane
4) Matrix
Early studies: sorting signal for targeting mitochondrial matrix proteins was in ____. The mitochondrial signal sequence was made of _____
-a stretch of N-terminal a.a residues
-positively charged a.a residues that aligned in one face of amphipathic helix
Mitochondrial proteins are synthesized where?
on free ribosomes
Requirements for import into mitochondria
- Membrane potential across the inner mitochondrial membrane.
- ATP hydrolysis
- molecular chaperones -> proteins of the hsp70 family that interact with mitochondrial proteins; maintain the mitochondrial proteins in an unfolded state and thus an import-component state.
-receptors on the surface of the mitochondria that interact with precursors + chaperones
Mitochondrial proteins imported into mitochondria first interact with a _____ at the surface of the outer membrane (_____). At some point before or during translocation the outer membrane complex engages an inner membrane complex (_____ ) to form contact sites at points of translocation.
-receptor complex
-the TOM complex
-the “TIM complex”
Mitochondria: translocation continues through this inner pore (_______) and the _____ go through a continuous cycle of _____ to segment of the polypeptide emerging from the channel
-the TIM complex
-mt-hsp70
-binding, releasing, and rebinding
As the polypeptide enters the matrix _____ is released in an ____-dependent manner and is replaced by another mitochondrial chaperone, _____, which assists in the _____
-mt-hsp70
-ATP
-mt-hsp60
-folding of a protein into its mature form
Proteins that are destined for the intermembrane space are translocated through the ______. The TOM complex and other complexes mediate the _____
-TOM complex directly into the intermembrane space
-direct integration of membrane proteins into the outer membrane
Kunkele paper showed two things: _____
-isolated TOM complex & characterized the pore
-reincorporated complex into membranes so they could view activity of channel
TOM complex major parts (4)
-TOM 70, TOM 40, TOM 20, TOM 22
TIM complex major parts (4)
-TIM 23/17, Pam17, mtHsp70, TIM 50
Ways to isolate TOM complex
-lyse cells
-separate mitochondrial membranes
-density centrifugation
how density centrifugation works?
-organelles have different sizes/density
-largest organelles, nucleus then mitochondria pellet first
-ER breaks into smaller vesicles -> microsomes
how to separate mitochondrial membranes?
-mitochondria in low osmotic solution swells and membrane breaks into vesicles
-differences in protein content in the membranes = differences in density
-density centrifugation separate OM from IM vesicles: OM vesicles are lighter, IM vesicles are heavier + sediment first
How Affinity Chromatography with Ni-NTA column works?
-protein tagged with C-terminal hexahistidinyl tag by modifying coding gene with homologous recombination
-C-terminal is the easiest place to add tag
-Histidines will bind to Ni column
-elution by imidazole
Membranes can be solublizied with detergents. Why? Types of detergents?
-to remove membranes (if you purify complex without removing membranes, membrane will be dragged with complex)
-non-denaturing detergent/non-ionic: digitonin, Triton-X-100
-denaturing detergent: SDS
using a mild detergent is better to ______
minimize the affect of detergent on protein function/structure
Gel filtration chromatography does what? descr.
-separate molecules based on molecular mass
-column of sephrose beads (porous) separate proteins based on size
-bigger proteins come out first; smaller proteins come out last (get stuck in beads)
Insertion of TOM complex how? measures what?
-added to black lipid membrane bilayer (useful for determining protein conduction channel -ex. ER)
-increasing insertion of channels into membrane by conductance, increase in conductance with increasing KCl
detergent binds to protein to _____
stabilizes hydrophobic part of membrane
detergent has critical micelle concentration which means that _____
micelles forms past this concentration
removing detergents removes _____
free lipids and micelles
intermembrane mitochondrial space protein
cytochrome C hemelyase (CCHL)
electron micrography involving staining with _____
uranyl acetate
How protease protection assay works?
-protein in bilayer by removing detergent
-add a soluble protease
-If the membrane protein is in the right orientation, the outside part will be cut off and the membrane part will be fine
-In the opposite orientation, that part of the protein will not be cleaved off (it’s protected)
specimen prep for cryogenic electron microscopy
-apply sample on holey carbon EM grid
-blot sample on filter paper
-plunge-freezing in lipid ethane (keeps things intact)
cryoEM: For single particle analysis (SPA) the images of randomly orientated homogeneous particles are recorded at ______. Involves multiple views of ______; this is why SPA is also known as _______
-low electron exposure (dose)
-many copies of the same molecule
-single particle averaging
SPA: each particle is in a different orientation, so they can be ______. The more orientations you image, _____
-pooled together to refine the structure
-the better
Kerscher paper summary
-Tim54p-Tim22p complex mediates insertion of proteins in IMM not translocation of proteins across inner membrane into matrix
-Tim 23p/Tim17p complex mediates transport into the matrix
MMMI is an _____ contact site
-ER-mitochondria
Tetrad dissection analysis steps
- Construction of disruption construct (gene is disrupted by the integration of a marker within it)
- Integration
- Tetrad dissection
- no growth = doesn’t have the essential gene
HA-tag expression on protein can _____
be used to purify/detect proteins
Osmotic shock leaves the ______. Trypsin is able to cleave anything in the _______. The ____ is still protected though.
-membrane full of holes
-outer membrane and in the intermembrane space
-matrix
A KO can grow if you ______
-add the essential gene back in with a plasmid
tim 54-1 mutant growth is rescued by ______ at 35ºC. tim23-1 mutant growth is rescued by ______.
-TIM22 and TIM54
-TIM17 and TIM23
TIM54-TIM22 complex does what?
import and integration of membrane proteins (multi membrane spanning protein)
TIM23-TIM17 complex does what?
import of matrix proteins and integration of single-pass membrane proteins
chloroplast is an organelle in ______ and came from ______
-photosynthetic organisms
-endosymbiotic theory
chloroplast function
energy interconversion by chemiosmotic mechanisms similar to mitochondria
Chloroplasts are also similarly organized to _____ but they also contain an additional membrane enclosed compartment which forms ______
-mitochondria
-disc-like sacs called thylakoid
differences between chloroplast and mitochondria
-chloroplast: uniform double membrane (no cristae)
-chloroplast is bigger than mitochondria (get damaged easily)
chloroplast vs mitochondria
-inner/outer membrane, inter-membrane space,DNA, ribosomes (C/M)
-matrix (M) + stroma (C)
-thylakoid space (C)
-thylakoid membrane (C)
thylakoid membrane of chloroplast is like _____
inner membrane of mitochondria (same activity)
membranes to pass through in mitochondria (4)
-outer membrane
-inter-membrane space
-inner membrane
-matrix
membranes to pass through in chloroplast (6)
-outer membrane
-inter-membrane space
-inner membrane
-stroma
-thylakoid membrane
-thylakoid lumen
The thylakoids are attached to one another such that ______. The thylakoid membranes appear almost as pitched off equivalents of _______.
-their lumens are interconnected
-mitochondria cristae
The inner membrane of chloroplasts does not fold into ____ and does not contain an _______.
-cristae
-electron – transport chain
The ______ are all contained in the thylakoid membrane.
-electron – transport chain
-photosynthetic (light-absorbing) system
-the ATP synthase
The _______ is the site of photosynthesis, the production of ATP and NADPH
-thylakoid membrane
Photosynthesis at chloroplasts
-Two linked but separate photo systems transfer of high energy electrons from chlorophyll to NADP+ (→ NADPH) and, in the process, creates an electro-chemical gradient which generates ATP.
-photosystem II then I transfer e- to NADP+ (from high redox potential to low)
-cytochrome complex pumps H+ into thylakoid space
-pH difference (H+ electrochemical gradient) of thylakoid membrane and stroma used to create ATP
-H+ comes from breakdown of H20 at cytochrome b6-f complex
thylakoid space pH? stroma pH?
-lower pH
-higher pH
Redox potential is a measure of the _______, which means that the more positive the redox potential, the _______, and the more negative the redox potential the more likely it is to ______
-ease with which a molecule will accept electrons
-more readily a molecule is reduced
-release electrons
________ creates H+ gradient across thylakoid membrane with an ↓ pH in thylakoid lumen
-H+ produced by breakdown of H20, b6-f complex and uptake by NADP+ → NADPH
ATP synthase (chloroplast vs mitochondria)
chloroplast: ATP synthase faces stroma from thylakoid membrane
mitochondria: ATP synthase faces matrix from inner membrane
chloroplast: The coupled photosystems accomplish the production of______ in the form of ______ and the production of _____
-energy
-ATP and NADPH
-02
chloroplast conducts several important “______” or ____ reactions.In these reactions ATP and NADPH are utilized in a carbon fixation cycle (termed the _____) to convert C02 to ______. It is an important intermediate for the production of carbon sources that are utilized for ______
-dark reactions
-carbon-fixation
-Calvin-Bensen cycle
-glyceraldehyde–3–phosphate
-energy production (glycolysis) and energy storage (as starch or sucrose)
An important enzyme in the calvin-bensen cycle is ____, which “fixes” CO2. Rbc is a _____ protein and constitutes over ____ the total chloroplast protein. Rbc is believed to be the most _____
-ribulose bisphosphate carboxylase (Rbc)
-stromal
-50 %
-abundant protein in nature
OM complex of chloroplast OM is called the ______ and the IM complex is called the _____.
-TOC complex
-TIC complex
Protein Import Into Chloroplast
- similar to mitochondrial import
-amino-terminal signal sequence similar to the mitochondria signal (problem: plants have both mitochondria and chloroplasts)
It has been shown that these signals are organelle specific by constructing _____. For example, replacing the signal as a mitochondrial protein with a chloroplast signal targets the protein
to the______.
-chimera proteins
-chloroplast
binding of precursor protein for chloroplast transport needs _____. translocation needs _____. In the stroma, precursor protein is _____
-low ATP
-high ATP
-cleaved off
TOC complex parts (4)
TOC75 (channel), TOC34, TOC159, TOC64
TIC complex parts (5)
TIC110 (channel), TIC40,TIC32,TIC55, TIC62
chloroplast experiment done in the dark to control ______, because in the light, the chloroplasts will _____
-ATP concentration
-generate ATP
TOC stands for _____
translocation channel of the outer chloroplast membrane- complex
Proteins can be denatured by ___
urea
