Cell Bio Exam 2 Guide

membrane bound proteins

proteins attached to the cytosolic face of the ER and are engaged in synthesis of secretory proteins

cytosolic free ribosomes

proteins not attached to any cellular membrane and are engaged in synthesis of non-secretory proteins

Smooth ER

specializes in synthesizing lipids

Rough ER

has a series of flattened spaces and acts as a binding site for ribosomes during translation → specifically when proteins are being synthesized

Microsomes

can be either rough or smooth. A homogenizer is utilized to break down rough and smooth ER into a mixture → can be separated using density centrifugation. smooth have LOW densities and stop sedimenting at LOW sucrose concentrations, while rough have HIGH densities and stop sedimenting at HIGH sucrose concentrations

Cotranslational translocation

a ribosome begins reading mRNA to make a protein, this new protein will obtain a N-terminal signal sequence, a helper component Signal Recognition Particle (SRP) helps carry this protein to the rough ER, the protein then docks on the ER and feeds into the membrane while its still being generated

Cotranslational translocation factors

SRP, SRP receptor in membrane, a translocon (in the membrane) that allows protein to feed through ER, and GTP hydrolysis

Cotranslational translocation for soluble secretory proteins

Ribosome begins reading mRNA and generating a protein while free floating in the cytoplasm, the generated signal sequence binds to the SRP and directs where to go on rough ER. Here the SRP receptor binds and the ribosome attaches to a translocon to feed the protein through. GTP hydrolysis occurs as SRP detaches, and signal peptidase cleaves the off the signal sequence

Cotranslational translocation in yeast

Translocon in Sec61a, the SRP receptor is Sec63complex, BiP is a cofactor that binds to ATP for activation and creates a complex that stabilizes the growing protein chain into the ER lumen

Direction for protein synthesis

N terminus → C terminus (N terminus comes out of the ribosome first)

Type I terminal sequence

The only type of signal sequence that will cut (ie. insulin receptor)

Type II terminal sequence

A non-cutting terminal sequence that requires a single internal hydrophobic anchor sequence → this sequence serves as the ER targeting signal and becomes the hydrophobic transmembrane spanning sequence. Consequently, the N terminal extends to the cytosol

Type III terminal sequence

a non-cutting terminal sequence that requires a single internal hydrophobic anchor sequence. The N terminal is in the ER lumen, and the C terminal is in the cytosol

Insertion of Tail-anchored proteins

a non-cutting terminal sequence that does not utilize SRP. The hydrophobic tail sequence acts as SRP - it needs ATPase to bind. The tail extends into the cytosol (meaning the N terminus is in the cytosol)

Type IV terminal sequence - multipass

a non-cutting terminal sequence. The first transmembrane segment is C terminal facing the cytosol. Each subsequent segment takes the opposite orientation as the one before - an even number of transmembrane anchored proteins will then mean that the N terminus and C terminus point in same directions

GPI Anchored Proteins

Composed of a fatty acid tail with an opposing polar end and it links to a protein → polar end connects to the C terminus of a protein in the ER lumen

Protein modifications before final destinations

glycosylation in the ER and Golgi (adding carbohydrate chains), formation of disulfide bonds to the ER, proper folding of polytide assembly to the subunits, and proteolytic cleavage in the ER, Golgi, and vesicle

Resident ER proteins

Proteins that aid in translocation and folding

Binding Protein (BiP)

the “chaperone” protein that aids in folding and multimeric assembly that prevents back sliding and unfolded protein response - also assists in translocation

Protein Disulfide Isomerase (PDI)

catalyzes oxidation of free SH groups to S-S bridges - helps proteins achieve correct folded structures

Sugars and Glycoproteins

____ on _____ get modified as they move through the Golgi

Endosome

Receives material that has entered the cell via endocytosis and decides where molecules should go

Lysosome

acidic organelles that receive materials intended for degradation - cell digestion

Dynamin

Required for clathrin coated vesicles → it pitches the neck of the bud and required GTP hydrolysis

S-S Formation and Rearrangement

the ionized S- of the substrate protein reacts with S-S bond in oxidized PDI to form and intermediate. Then the second ionized S- of the substrate protein reacts with intermediate to form a S-S within the protein

Glycosyltransferases

An enzyme that adds carbohydrate chains attached to the -OH in serine and threonine OR carbohydrate chains to the amide nitrogen (N2H) of asparagine

Biosynthesis of N-linked Oligosaccharaide precursor

the building of a large sugar chain that will attach to the protein in the ER - 3x glucose, 9x mannose, and 2x N-acetylglucosamine → all of it is assembled on dolichol phosphate and gets flipped to ER lumen

Protein glycosylation (N-linked oligosaccharide) in the RER

once biosynthesis of precursor chain is assembled - three glucose present signal this, the ER trims its designated components, and is then transported to the cis-Golgi

Unfolded Protein response

Ire1 monomers form dimers. This causes endonuclease activity to splice Hac1 mRNA. That spliced Hac1 mRNA is translated and taken back to the nucleus

Modification of N-linked Oligosaccharides

Used to monitor folding and quality control. One glucose is retained to bind to CNX/CRT. To retain the misfolded protein, UGGT can add a glucose and reflow binding. However if the protein cannot be folded, ERa-mannosidase will reduce mannose residue and bind the protein to OS-9 for degradation

Calnexin and Calreticulin

components in protein folding that bind to N-linked oliggosaccharaides and prevent incorrect folding with adjacent proteins

Coated vesicles

Compartments that transport proteins between cellular compartments. Their coat proteins (COPI, COPII, or clathrin) help select cargo and direct vesicle trafficking to specific destinations

COPII

vesicle coat type that transports proteins FROM the ER to the cis-Golgi (Sar1) → Sar1 serves as binding site for the coat proteins, after the coat is complete, detachment of coat occurs. Signal sequence is Di-acidic (Asp-X-Glu)

COPI

vesicle coat type that transports proteins FROM the cis-Golgi, to the ER, and later to the Golgi cisternae. signal sequence is KEDEL and GTPase is ARF

Cathrin (and adapter proteins)

vesicle coat type that transports proteins FROM the trans-Golgi TO the endosome (ARF)

Rab GTPase

Small GTP-binding proteins that regulate vesicle targeting and docking. In their active GTP-bound state, they recruit tethering proteins that guide transport vesicles to the correct target membrane. This means v-SNARE proteins attach, formulate the SNARE complex with t-SNARE proteins on target membrane, and assimilate membrane fusion

Anterograde Transport by COPII

Forward vesicle transport from the ER to the cis-Golgi → requires di-acidic sorting signal for cargo protein

Retrograde Transport by COPI

Reverse transport from the cis-Golgi to the ER for particles that need to be returned. It retrieves v-SNAREs and other misc. items. The high pH in the golgi promote binding between KEDL and its receptor → it is taken back to a lower pH in the ER where the binding detatches

M6P

a carbohydrate marker added to lysosomal enzymes in the cis-Golgi apparatus. It is recognized by M6P receptors in the trans-Golgi network, which package the enzymes into clathrin-coated vesicles for transport to endosomes and ultimately lysosomes

Endocytosis

Receiving lysosomal enzymes from outside of the cell to the late endoscope (low pH) - involves budding, coating, and disassembly processes

Transocytosis

Involves endocytosis from blasolateral membrane and moves and fuses to the apical membrane (ie. move materials from one side to another side)

Internalization

Cells uptake macromolecules from surrounding outside intracellular destinations

Cell Polarity

ability of a cell to generate functionally distinct regions within itself

Cytoskeleton

composed of microfilaments, microtubules, and intermediate filaments

Microfilaments

rigid, high dynamic, and polarized protein fiber network of actin-binding proteins - motor protein is myosin → actin binds to ATP

Microtubules

rigid, high dynamic, and polarized tube shaped structures of tubulin → motor proteins are kinesis and dyneins

Intermediate Filaments

strong, less dynamic, and non polarized type of cytoskeletal structure protein that provides mechanical strength and structure to cells and tissues → composed of lamins and DO NOT have motor proteins

Critical Concentration (Cc)

the minimal concentration of monomers at which filaments form → when added short stable actin filaments as the starting point, elongation happens immediately

Microtubule Dynamics

Bulk tubulin versus individual microtubulin → bulk has its concentration above the critical point and polymerization assembly occurs while dynamic grows and shrinks overtime

Formats of actin

G-actin and F-actin

Dynamics of actin

nucleation, elongation, and steady state

Profilin

Binds to G-actin on the side opposite of the nucleotide binding cleft → binds to the (+) end and promotes filament growth by adding actin monomers to it

Cofilin

Binds to ADP-actin subunits with F-actin on the (-) end→ promotes depolymerization of filaments and actin disassembly - induces a small twist and allows more free (-) ends

CapZ

an actin capping protein that binds to the positive (+) end and prevents filament growth

Tropomodulin

an actin-capping protein that binds to the minus (−) end and prevents filament growth or shrinkage at that end

Thymosin-B4

STores actin filaments that cannot be added onto another monomer (buffer)

Arp 2/3 Complex

Creates a starting point for branched filament assembly. Nucleation promoting factors (NPF) bind to actin via WH2. This complex then changes its conformation as it binds to 2 more NPFs. After start is initiated NPF is released. This mechanism grows from the (+) end until it meets Zap (stop codon)

Listeria Monocytogenes

A bacteria obtaining ActA. This activates the host cell's Arp2/3 complex - since Arp2/3 generates branched actin filaments, it propells the bacterium through the cytoplasm and enables the cell to spread

Myosin

Motor protein for actin filaments → the head domain interacts with actin filaments (S1 = the head). The distance filaments travel depends on their neck lengths

Class I Myosin

Step size is 10-14 nm, functions in membrane association + endocytosis, and moves in the (+) direction towards the barbed end

Class II Myosin

A (+) end directed actin motor protein with two motor heads and a long coiled-coil tail. Its step size is 8nm and it generates force for muscle contraction and cellular contractility

Class V Myosin

Step size is 36 nm, and it uses two motor heads to transport vesicles along actin filaments (cunty motor protein edit) for organelle support