Cell Biology Exam 3

Herbicides

-killing weeds can be done by inhibiting their electron transport (shutting down photosynthesis)

-common herbicides bind to a core protein of PSII

-light reactions serve as targets of herbicides

-some herbicides produce O2 radicals, which are toxic to human tissue → cancer

Photophosphorylation

-the machinery for ATP synthesis in a chloroplast is similar to that of mitochondrial enzymes

-the ATP synthase sonsists of a head (CF1) and a base (CF0)

-the CF1 heads project outward into the stroma, keeping with the orientation of the proton gradient

-protons moves into the lumen through the CF0 base of the synthase

-protons move through the CF0 base of the synthase

-measurements of chloroplasts during ATP synthesis show an increase in ^pH (the only energy source in chloroplasts, no electrical difference)

-the movement of protons during photosynthesis does not create a significant change in the membrane potential since other ions are transported simultaneously

Noncyclic photophosphorylation

-the movement of electrins (from water → NADPH) during the formation of oxygen; ions move in a linear path

Cyclic photophosphorylation

-carried out by PSI independently of PSII; “boosting- passing back- pumping H+”, → H+ to ATP synthase → extra ATP

• provides additional ATP required for carbohydrate synthesis

-equal concentration of ATP & NADPH, but to make one carbon sugars extra ATP is needed

• when more ATP is needed, ferredoxin passes electrons back to cytochrome b6f → 18 ATP + 12 NADPH

CO2 fixation and carbohydrate synthesis

-the movement of carbon in a cell can be followed during photosynthesis using [14 C]O2 as a tracer

-extracts of cells are then analyzed by autoradiography by identifying radiolabeled compounds compares to known standards

• chromatogram from algal cells after incubation with [14C]O2 showing accumulations of 3-phosphoglycerate

  • conclusion: PGA is the 1st band of intermediates from the Calvinc cycle

Carbohydrate synthesis on C3 plants

-C3 plants produce a 3 carbon intermediate, PGA

• CO2 is condensed with RuBP to form a 6 carbon molecules which splits into 2 molecules of PGA

• the condensation of RuBO and the splitting of the 6 carbon molecule are catalyzed by RUBISCO

  • RUBISCO is the most abundant protein on Earth and has a very low turnover number

  • RUBISCO fixes ~ 3 molecules of CO2/sec

Calvin Cycle (C3 Pathway)

1. carboxylation of RuBO to form PGA

2. reduction of PGA → G3P (product of the calvin cycle) using NADPH & ATP from light reactions

3. regeneration of RuBP (where the extra 6 ATP is spent)

-for every 6 molecules of CO2 fixed, 1 molecules of GAP are produced

• the GAP molecules can be exported into the cytosol and used to synthesis sucrose

• GAP can also remain in the chloroplast where it is converted to starch

Sucrose

the organic building block in plants (similar to glucose in animals)

Starch

stored in the plant’s leaves and provides it with sugars at night (similar to glycogen in animals)

Redox control

-a light dependent regulator of chloroplast metabolism

-thioredoxin can have 2 forms:

1. reduced → calvin cycle enzyme of activation → synthesis of carbohydrates in chloroplast (need sunlight to activate calvin cycle enzymes)

2. oxidized → occurs in the dark and causes enzyme inactivation

Photorespiration

-series of reactions that involve the uptake of O2 and release of CO2

-it accounts for the loss of up to 50% of fixed Co2

-RUBISCO also catalyzes the attachment of O2 to RuBP → 2-phosphoglycerate

• bad process for plants

-glycolate is then transferred to the peroxisome and leads to the release of CO2

Peroxisomes & Photorespiration

-glycolate is shuttled out of the chloroplast into a peroxisomes

• in peroxisomes: glycolate → glyoxylate → glycine

• in the mitochondrion: 2 glycine → 1 serine + release of 1 CO2

C4 pathways

-C4 pathways involves the production of phosphoenolpyruvate (PEP), which then combines with Co2 to produce 4 carbon compounds oxaloacetate or malate

• plants using this pathway are C4 plants (tropical grasses)

• C4 plants move chloroplasts inward and cut off Co2 close to the chloroplasts

-in a hot, dry environment, C4 plants get enough CO2 for photosynthesis while keeping their stomata partially closed to prevent H2O loss

-point of C4 pathways is to concentrate CO2 at the chloroplast so Rubisco can add CO2 (and not O2) to RuBP

-C4 plants separate the entry and exit of CO2 by space

CAM plants

-carry out light reactions and CO2 fixation at different times of the day using PEP carboxylase

-keep their stomata closed during the day (to prevent CO2 from coming in) and open at night

-the malate generated in mesophyll cells is transported into the cell’s central vacuole

-during the day (stomata closed) → malic acid is moved into the cytoplasm

• in the cytoplasm, malic acid can be fixed by Rubisco under low O2 concentration conditions

• carbs are then synthesized using energy from ATP & NADPH generated in the light-dependent reactions

-CAM plants separate entry and exit of CO2 by time

Genetic systems of mitochondria and chloroplasts

-resemble the systems of prokaryotes

-over time, mitochondria and chloroplasts have exported most of their genes to the nucleus by gene transfer

-mitochondria have a relaxed codon usage and can have a variant genetic code

-chloroplast and bacteria share many striking similarities

-organellar genes are maternally inherited in animals and plants

-mitochondria:

• 13 proteins in humans

• 2 rRNAs and 22 tRNAs

• molecule (mtDNA), ribosomes, and enzymes; RNA and proteins can be synthesized in the matrix

Endomembrane system

-a coordinate unit of membrane-bound compartments

• ER

• Golgi complex

• endosomes

• lysoomes

• vesicles

-materials packaged in small, membrane-bounded transport vesicles

• bud from a donor membrane compartment → move via motor proteins on microtubules and microfilaments of the cytoskeleton → fused with the membrane of the acceptor compartment

Biosynthetic pathway

proteins are synthesized in the ER, modified at the golgi, and transported throughout

Secretory pathway

protein synthesized in the ER are discharged from the cell (via vesicles)

Endocytic pathway

-materials move from the outer cell surface → compartments via endosomes & lysosomes

-ex: endocytosis, exocytosis, phagocytosis

Constitutive secretion

-materials are transported in secretory vesicles and discharged in a continual manner

-always producing and always releasing (unregulated secretion)

Regulated secretion

-materials are stored in vesicles and discharged in response to a stimulus, ex: insulin

• RS occurs in endocrine cells (hormones), digestive enzymes, neurotransmitters

• secreted materials can be stored in large, densely, packed secretory granules

-sorting signals: used to route cargo to their designated cellular location; encoded in the amino acid sequence of the proteins or in the attached oligosaccharides

Autoradiography

-a method to visualize biochemical processes using radiolabeled materials

• used to determine where secretory proteins are synthesized by using labeled amino acids

-pulse chase experiments: pulse labeling cells with radioactive amino acids, then allowing the cells to function normally to visualize the synthesis and transport of secretory proteins

GFP-based protein tracking

-green fluorescent protein (GFP): small protein which emits green fluorescent light

-fusing genes to GFP allows the study of protein traffick

• may change protein function

• may lead to overexpression of protein

Biochemical analysis of subcellular fractions

-subcellular fractions: homogenize cells and isolate some organelles

• can then be separated from one another- purified smooth OR rough ER (subcellular fractionation)

-microsomes: membrane vesicles derived from the endomembrane system

Insights gained from the use of cell-free systems

-liposomes

-buds and vesicles can be produced when purified proteins normally on the cytosolic surface of transport vesicles in the cell are added

• can study proteins: 1. that bind to the membrane to initiate vesicle formation, 2. those responsible for cargo selection, 3. those that sever the vesicle from the donor

Insight gained from the study of mutant phenotypes

-screen for mutant yeast cells that exhibit an abnormal distribution of cytoplasmic membranes reveal proteins that function in secretion

• results: mutating proteins involved in secretion 1. made the rough ER grow to capacity (unable to release vesicles) 2. golgi with several vesicles that cannot fuse and deliver cargo

-RNA interference (RNAi) is used to inhibit mRNA translation into proteins

• can “knock down” the expression of certain proteins OR inhibit the mRNA and observe the effect on proteins

• using siRNAs, one can find genes involved in various steps of the secretory pathway

ER

-network of membranes that penetrates much of the cytoplasm and has a lumen separated from the cytosol by the ER membrane

-highly dynamic structure, 2 compartments share some proteins and activities

-RER (rough ER):

• ribosomes bound to cytosolic surface

• flattened sac (cisternae) connected to itself

• continuous with the outer membrane of the nuclear envelope

-SER:

• lacks ribosomes

• membranes are highly curved and tubular

• continuous with RER

Smooth Er

SER functions include:

1. steroid hormone synthesis in endocrine cells of the gonad and adrenal cortex (making lipids)

2. detoxification of organic compounds in the liver via oxygenases including the cytochrome p450 family (detoxification of enzymes, like alcohol)

3. calcium ion sequestration and regulated release (Ca 2+ storage in muscle)

Rough ER

-the nucleus and RER are near the basal surface, facing the blood supply

-the RER is the starting point of the biosynthetic pathway for secretory proteins

-about 1/3 of the proteins are synthesized at the RER and released into the ER lumen in a process called co-translational translocation

-remaining polypeptides synthesized on “free” ribosomes in the cytosol

• ex: goblet cells are specialized cells that produce mucus (aka glycoproteins) and deposits it at the top of the epithelium

Membrane bound vs. free ribosomes

-free ribosomes: found floating in the cytosol; make enzymes for glycolysis; translation starts with free ribosomes in the cytosol

-the site of protein synthesis is determined by the sequence of amino acids in the N-terminus portion of the polypeptide

-secretory proteins contain a signal sequence at their N-terminus that directs the emerging polypeptide and ribosome to the ER membrane

-transmembrane proteins made at rough ER + secreted proteins (ex: insulin)

-the polypeptide moves into the cisternal space of the ER through a protein-lined, aqueous channel in the ER membrane, as it is being synthesized (at the RER)

Synthesis of secretory, lysosomal, or plant vacuolar proteins

-co-translational translocation deposits protein into the ER lumen

-polypeptide signal sequence: 6-15 hydrophobic amino acid residues targeting polypeptides to the ER membrane

-several of the steps of the synthesis and trafficking of secretory proteins are regulated by the binding or hydrolysis of GTP

-SRP and SRP receptor are G proteins that interact with one another in their GTP-bound states; GTP hydrolysis triggers the release of the signal sequence by the SRP

Steps of secretory/lysosomal/plant protein synthesis

1. signal sequence recognized by signal recognition particle (SRP)

2. SRP binds polypeptides and ribosome, which pauses translation

3. complex is recruited to the ER membrane through interactions between the SRP and the SRP receptor on the ER membrane

4. ribosome is handed to the translocon

5. protein is threaded through the translocon into the ER lumen → SRP detaches → signal sequence is chopped off by signal peptidase

6. translation continues

-translocon: a protein channel embedded in the ER membrane

RER: processing newly synthesized protein

-signal peptidase: removes the signal peptide from nascent polypeptide

• occurs while carbohydrates are added by oligosaccharyltransferase

-the ER membrane provides a large surface area for ribosomes to attach, and the lumen gives a specialized local environment that favors protein processing

RER: synthesis of integral membrane proteins

-integral membrane proteins are synthesized co-translationally, and their hydrophobic transmembrane segments are shunted from the translocon into the lipid bilayer

• normal orientation: N-terminus on the inside, C-terminus in the cytosol

-during membrane protein synthesis, the inner lining of the translocon orients the nascent polypeptide so that the more positive end faces the cytosol

• orientation flips: N-terminus in the cytosol, C-terminus inside

-there are groups of charges in the translocon because of the amino acids: negative charge on top, positive charge on bottom

-attraction of opposite charges is what holds the protein in the hydrophobic portion of the membrane

-depending on the amino acid sequence of the protein and charge, the translocon can shift the nascent polypeptide to flip orientation so opposite charges can attract

Multispanning proteins

-sequential segments typically have negative orientations, so their arrangement in the membrane is determined by the direction of the 1st segment

Tail anchored proteins

-lack a signal sequence, but are synthesized in the cytoplasm, are targeted to the ER through interactions with proteins in the “Get” proteins

-fully synthesized in cytosol, then moved to ER

Membrane biosynthesis

-membranes arise from pre-existing membranes

-membranes are enzymatically modified as they move from ER into other cellular compartments; orientation is maintained

-membranes are asymmetric with a cytosolic face and a luminal/extracellular face established in the ER; symmetry is maintained

-the glycosylated end of the protein moves to the exterior of the membrane → forms glycocalyx

Lipid composition of membranes

-membranes of different organelles have different lipid compositions; they can change their lipid composition by:

• using lipid-modifying enzymes to convert a phospholipid to another (changing polar head groups via enzymes)

• preferentially including or excluding phospholipid vesicles (shifting lipids laterally to concentrate the lipids wanted)

• exchanging lipids between organellar compartments using lipid transfer proteins

RER: glycosylation

-nearly all proteins produced on RER become glycoproteins

-addition of sugars to an oligiosaccharide is catalyzed by glycosyltransferases, each transfers a specific monosaccharide to the growing end of the carbohydrate chain

-the sugar arrangement in the oligosaccharide chains of a glycoprotein depends on the spatial localization of enzymes in the assembly line

-dolichol: will be inserted into the membrane of the RER (and phosphorylated)

• then sugars are added to the dolichol; 2 NAG is added, then mannose is added to the dolichol out in the cytosol

• dolichol is flipped (requiring energy); sugars are now inside the ER lumen → more mannose is added to create a 3-pronged structure → then 3 glucoses are added to 1 prong of mannose → oligotransferase removes all the sugars (2 NAG, 3 glucose, mannose) and attaches them to nitrogen = N-linked glycosylation

Importance of N-linked glycosylation

contributes to glycocalyx + plays a role in proteins folding up (provides the sugars used by chaperones to fold proteins)

Quailty control for proteins

-a glycoproteins goes through a system of quality control to determine fitness for UGGT; ensures that misfolded proteins do not move forward

-misfolded proteins will be glucose tagged, mannose deficient, and ultimately degraded by proteasomes (ER-associated degradation)

Glucosidase

-cut off glucoses

Calnexin

-a chaperone that helps proteins fold correctly

• after proper folding, the 3rd glucose is cut off and the protein exits the RER via exocytosis in a vesicle to the Golgi

• misfolding: 3rd glucose is cut off and UGGT adds the glucose back → protein goes back to calnexin… cycle restarts

  • proteins that NEVER fold up, have enzymes cut off the mannoses (slow-acting mannosidase) → leaves the RER without a vesicle → proteasome in cytosol → degraded into amino acids

RER: Destruction mechanisms of misfolded proteins

-accumulation of misfolded proteins → unfolded protein response (UPR)

-sensors in the ER are kept inactive by the chaperone BiP

-when misfolded proteins accumulate, BiP is capable of inhibiting the sensors

-activated sensors send signals to trigger proteins involved in destruction of misfolded proteins

-members of the URO can:

• activate production of proteins that shut down ribosomes (stop protein synthesis)

• make machinery for proteasomes

Key concepts of Fultz story

-(transmissible) spongiform encephalopathies: scrapie, kuru, mad cow

• all showed evidence of microscopic holes in the brain

-every single transmissible disease was related to a nucleic acid

-treatment of infected mice with proteases stop the spread of illness → infected proteins (prions): have a different shape and are denature resistant

ER → Golgi vesicular transport

-1st step of vesicular transport

-RER have specialized exit sites where transport vesicles are formed (no ribosomes)

-transport vesicles fuse with one another and form the ERGIC (ER golgi intermediate complex) toward the golgi complex

Golgi complex

-a stack of flattened cisternae

• cis face of Golgi faces the ER: receives signals, where vesicles from the ER come to fuse with the Golgi

• trans face is on the opposite side of the stack: leaving the Golgi

-trans golgi network (TGN): sorts proteins to the pm or various intracellular destinations

trans cisternae

medial cisternae

cis cisternae

-cis golgi network (CGN): sorts proteins for the ER or the next golgi station

Golgi complex function

-assembly of carbohydrates found in glycolipids and glycoproteins takes place in the golgi

-sequence of incorporation of sugars into oligosaccharides is determined by glycosyltransferases

Vesicular transport model

-cargo is shuttled from CGN → TGN in vesicles

Cisternal maturation model

-each cistern “matures” as it moves from the cis face to the trans face

• current model: similar to CMM, but with vesicle retrograde transport; golgi cisternae serve as primary anterograde carriers

• favored model

Protein coats

-materials are carried between compartments using coated vesicles

-protein coats have 2 functions:

• cause the membrane to curve and form a vesicle

• select the components to be carried by vesicle

3 types of vesicles

-COPII-coated vesicles: move materials from the ER “forward” to the ERGIC intermediate compartment and golgi complex

-COPI-coated vesicles: move materials from ERGIC and golgi “backward” to the ER, or from trans golgi → cis golgi

-clatherin-coated vesicles: move materials from the TGN to endosomes, lysosomes, and plant vacuoles

COPII-coated vesicles

-bud off specialized domains of the ER, ER exit sites (ERESs); start of biosynthetic pathway

-ER export signals found in cytosolic tails of transported proteins

-COPII coats select and concentrate proteins/enzymes that are going to the golgi

-Sar1: exchanges GDP for GTP and changes shape to insert itself into the PM

-sec 23 and sec 24: bind to the PM and begin to curve the membrane

• sec 24 binds to cargo receptors bound to our cargo

-sec 13 and sec 31: form the outer coat of the coated vesicle

COPI-coated vesicles

-COPI vesicles always move backwards (retrograde)

1. movement of golgi resident enzymes back to their layer

2. movement of ER resident enzymes from the ERGIC and golgi back to the ER

-organellar proteins are maintained by:

1. retention of resident molecules excluded from transport vesicles

2. retrieval of “escaped” molecules back to normal compartment

-retrieval signals: at the c-terminus of amino acid sequences found in resident ER proteins, ex: KDEL

Sorting proteins at the TGN

-sorting and transport of lysosomal enzymes utilizes clatherin-coated vesicles

-lysosomal proteins are tagged in the cis golgi with phosphorylated mannose residues

-tagged lysosomal enzymes are recognized and captured by mannose 6-phosphate receptors (MPRs), which are bound by coat proteins

Sorting and transport of lysosomal enzymes

-clatherin coded vesicles contain:

• Arf-1: has a GGA adaptor attached; GGA adaptor binds to the receptor for MPR and binds to clatherin

• Arf-1-GTP is used to initiate membrane curvature and recruit adaptors

Targeting vesicles to a particular compartment

-Rabs: family of small G-proteins, which cycle between an active GTP bound state and an inactive GDP bound state

-GTP-bound Rabs associate with membranes by a lipid anchor

-SNARE proteins: integral proteins that bring vesicle and target in contact

• v-SNARE: always in the vesicle

• t-SNARE: always on the target membrane

-the v-SNAREs and t-SNAREs bind to each other and start twisting together → pulls membranes together → membranes fuse

Exocytosis

-discharge of a secretory vesicle a granules after fusion with PM

-process is triggered by an increase in [Ca 2+]

-luminal side of vesicle becomes the outer surface of the PM and the cytosolic side becomes the inner surface (cytosol remains cytosol)