Features of the Cell Membrane and Trafficking Flashcards
Metacognition
Thinking about thinking
Cytoplasm
Everything within cell outside nucleus including organelles and cytosol
Cytosol
Gel like substance composed of mostly water, ions, and proteins. pH is ~7.4. High concentrations of glutathione which acts as redox buffer.
Cell/Plasma/Cytoplasmic Membrane
Membrane made of pure phospholipids is semi-permeable. Constructed from amphiphilic lipids (fatty acids, oils, steroids-water insoluble).
Phospholipid
Hydrophobic tails(kink in one to allow for cylindrical shape). Hydrophobic head with Glycerol/sphingosine base, phosphate, and variable top regions.
Passive Transport
Requires no energy. Channel-mediated, transporter-mediated, or simple diffusion.
Lipid Bilayer
Phospholipids pack together in energetically favorable form (Hydrophobic interior and hydrophilic exterior).
Passes Through Fast: Non-polar (Fat-soluble live estrogen), gases(like O2 or CO2)
Passes Through Slow: Small Uncharged Polar (like water sometimes, ethanol, glycerol, acetic acid at pH 1.5)
Doesn’t Pass Through: Large, polar, charged (like ions, sucrose, acetic acid at pH 10)
Passive Transport
Requires no energy. Channel-mediated, transporter-mediated, or simple diffusion. Utilizes concentration gradient with no membrane potential or electrochemical gradient with membrane potential.
Active Transport
Requires Energy. Works against electrochemical or concentration gradients.
Diffusion
Passively right through the membrane. Diffusion is the net movement of anything from a region of higher concentration to a region of lower concentration. Diffusion is driven by a gradient in concentration.
FRAP
Fluorescence recovery after photobleaching . Bleach and see how long it takes to recover light. Quantify lateral diffusion.
Signal Hypotheis
Proposes that proteins encore sorting motifs. Proteins that leave the cytosol have intrinsic signals that direct them to the appropriate organelle.
Sorting Signal
Coded by protein itself, cis-acting (within/part of the protein). Recognized by Sorting Receptor (trans-acting: something else that can recognize signal) like Signal Recognition Particle (SRP) which recognizes signal as it emerges from ribosome. Binding causes a quick pause in translation. Translation directed to ER. Import Mechanism also involved.
Necessary
A necessary condition is a condition that must be present for an event to occur. Can protein do it with or without it?
Sufficient
A sufficient condition is a condition or set of conditions that will produce the event. Is protein enough to cause it to happen?
Karyopherins (KAPS)
Major Family of nuclear import and export receptors. Similar but different specificities for NLS & NES.
Importins
A type of karyopherin that transports protein molecules into the nucleus by binding to NLS. It docks at NPC, moves through, and releases cargo when interacting RanGTP and recycles receptor.
Exportins
Exportins bind to proteins with NES in association with ranGTP and dock at NPC. Then it passes into the cytoplasm through the NPC. The protein cargo is released when the GTP is hydrolysed and the exportins diffuse back into the nucleus.NTF2 Mediates nuclear import of Ran.
Nuclear Localization Signal (NLS)
Amino acid sequence that ‘tags’ a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface.
Nuclear Export Sequence (NES)
A nuclear export signal (NES) is a short target peptide containing 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport.
Gated Transport
Gated transport is most notable as the protein traffic between the cytosol and nucleus occurs between topologically equivalent spaces, which are in continuity through the nuclear pore complexes. The nuclear pore complexes function as selective gates, where selected macromolecules are actively transported while smaller molecules are allowed free passage.
Nuclear Pore Complex (NPC)
Have conserved structure with 8-fold symmetry. Composed of NPC proteins (nucleoporins). Have nucleoplamic face “baskets” and cytoplasmic face “filaments”.
Inner Nuclear Membrane
Lipid and protein compositions differ with outer membrane. Lamins for the nuclear lamina inside.
Outer Nuclear Membrane
The outer nuclear membrane is continuous with the endoplasmic reticulum membrane and internuclear membrane.
Perinuclear Space
The space between the nuclear membranes
Small GTPases/Small GTP Binding Proteins
Used to witch trafficking signal on and off. GDI=GDP dissociation inhibitor. GDI binds smallGTPase-GDP form and brings it away from lipid bilayer.
GTPase Activating Protein (GAP)
Promotes the conversion of GTP to GDP by increasing small GTPase activity .
GTP Exchange Factor (GEF)
Exchanges GDP for GTP.
RAN
Nuclear import and export. RanGTP enriched in nucleus and RanGDP enriched in cytoplasm. Gives directionality to transport.
Immunofluorescence
See protein of interest. Label multiple proteins with multiple colors. You can live image! May interfere with normal function because large! Not always easy to tag with fluorescence.Add antibodies that are fluorescently labeled. Kills cells. Use multiple antibodies to amplify. Also because it’s hard to have Fluorescein specific for an antibody.
Green Fluorescent Protein (GFP)
Very bulky. Can use to tag proteins and view live. Make proteins easily visible.
Signal Sequence
Usually refers to ER signal sequence. It is hydrophobic. The signal sequence at N-terminal of protein is recognized by SRP as it emerges from ribosome. It is later cleaved off by Signal Peptidase. It then laterally diffuses out of the Sec61 channel and is later degraded.
Translocation
Protein is inserted into ER.
Conformation Change
Change in the shape of a macromoleculed
Secretory Pathway/Exocytosis
ER->Golgi ->Secretory Vesicles->Destination (Plasma Membrane)
Endoplasmic Reticulum
Gateway to secretory pathway. Smooth and Rough. Also the storage site of calcium(Sarcoplasmic reticulum)
Rough ER
Protein translocation to create properly folded luminal and transmembrane proteins and glycoproteins
Smooth ER
Site of lipid synthesis and membrane biogenesis
Co-translational translocation
Translation occurs as it is inserted into the ER.
Signal Recognition Particle (SRP)
Recognizes and binds signal sequence and brings protein/ribosome complex to Sec61 translocon.
SRP Receptor
In rough ER and binds to SRP so that ribosome can then translocate protein.
Sec61 Complex/Translocon
Water filled pore. It stays open because proteins bind, changing the conformation and the insertion of hydrophobic signal sequence. Associates with ribosome and serves as entry for protein.
Signal Peptidase
Cleaves Signal sequence so that it may laterally diffuse.
Post-translational Translocation
Requires BiP and ATP. Can happen but needs different proteins. Translation on free-ribosome.
BiP
Chaperone protein that binds unfolded proteins as they are translocated into ER. They protect proteins when they are in the process of folding, shielding them from other proteins that might bind and hinder the process. Post-translational insertion requires BiP and ATP.
Chaperone
Proteins that help with correct folding
Luminal
Luminal or Extracellular space (Inside organelles/outside cell)
Signal-Pass Transmembrane Protein
Need about 20 amino acids to pass through membrane in a simple alpha helix. Can have a signal sequence(a start-transfer sequence) or an internal signal sequence)
Multi-Pass Transmembrane Protein
Goes through multiple times. Multiple start/stop transfer sequences.
Start-Transfer Sequence
Hydrophobic sequences.
Stop-Transfer Sequence
Hydrophobic sequence and becomes transmembrane domain of protein.
Hydropathy Plot/Hydropathy Index
Plus means hydrophobic (transmembrane region). Must be at least 20 amino acids in length. Count peaks!
Positive Inside Rule
Cytosol is negative (phosphotidyl-serines). ER Lumen is positive.
Topology/Topological Signals
Different topologies produced by signal-sequences, anchors, modifications, glycosylated
Protein Disulfide Isomerase (PDI)
Catalyzes sulfide bonds to form between cysteine in proteins in ER. Assist with folding in ER especially.
Glycosyltransferase
Enzymes that catalyze the addition of sugar to protein in ER especially.
N-linked Glycosylation
Occurs in ER. Oligosaccharide added to asparagine (ASN)
GPI-Link
Lipid anchor added in the ER to protein to keep protein bound to membrane. Temporary C-terminal stop-transfer sequence holds protein until GPI-link is formed
Unfolded Protein Response (UPR)
Feedback Mechanism to help cope with misfolded proteins. Stimulated by unfolded proteins. IRE1, PERK, and ATF6
IRE-1
Misfolded proeins activate kinase, self-phosphorilation and dimerization. Endoribonuclease activity is activated and removes intron sin RNA that leads to transcription regulator or ER chaperones that then help fold proteins.
Endocytosis
Substances are brought into the cell. Includes pinocytosis and phagocytosis. Recycle receptors, nutrients, glucose, viruses, neurotransmitters.
Exocytosis/Secretion
Cell transports molecules out of the cell by secreting them through pathway. Growth factors, immunoglobulins, hormones, waste, neurotransmitters.
Golgi
It acts to process and package the macromolecules such as proteins and lipids that are synthesized by the cell. Tubular-vesicular network with cis Face (Close to ER and nucleus) and trans Face (close to plasma membrane). It has distinct sub domains with their own chemicals that modify proteins they see. Extensive remodeling of glycosylation motifs occur in Golgi.
Early Endosome
Come right from membrane endocytosis. Will mature or recycle. Multiple come together to form multi-vesicular body (a type of late endosome).
Recycling Endosome
Recycle to plasma membrane instead of being degraded.
Late Endosome
Will fuse with lysosome to form endolysosome to be degraded .
Lysosome
Containing degradative enzymes enclosed in a membrane. Can fuse with endosome to form endolysosome.
Vesicle
Proteins move from place to place via membrane-enclosed transport vesicles.
Arf
Arf GTPases initiate COP1 and clathrin coat formation at Golgi (Comparable to Sar-1).
Sec23/24
SAR1-GTP recruits Sec23 which binds to Sar1GTP and then recruits Sec24. Sec24 binds to receptors that recognize cargo and recruit Sec13/31.
COPII
Involved in transport between ER to Golgi.
Dynamin
Mediates vesicle scission for clathrin coated vesicles. Forms a helix structure around and GTPase domain of dynamic uses GTP hydrolysis to change conformation.
T-SNARE
On membrane. Fuse and lock with V-SNARE during docking so that vesicle can fuse. Membranes must be within 1.5nm of each other.
V-SNARE
On vesicle. Fuse and lock with T-SNARE during docking so that vesicle can fuse.Membranes must be within 1.5nm of each other.
Rabs
RabGTPases guide vesicles to targets and mediate steps in vesicle transport. Interact with Rab effector protein during tethering. GDI (G-protein dissociation inhibitor) binds RabGDP and keeps it away from membrane.
NSF
Uses ATP and accessory proteins to unwind SNARES .
V-SNARE
On vesicle. Fuse and lock with T-SNARE during docking so that vesicle can fuse. Membranes must be within 1.5nm of each other.
EndoH
EndoH resistance marks entrance into medial Golgi. Early in Golgi, endow can remove sugars, later on it can’t.
Pulse Chase
Label proteins with radioactivity and then add unlabeled amino acids after to track proteins through secretory process.
KDEL
a sorting signal for retrieval to the ER. Other signals include KKXX.
KDEL-Receptor
If it recognizes by KDEL signal, will bud off and be sent back.
AP2
Clathrin adaptor protein that recognizes endocytic motifs on cargo proteins. Recruits clathrin to come form vesicle. Works on the cell membrane to internalize cargo in clathrin-mediated endocytosis.
