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Flashcards in Mindterm 1 Deck (74):

Describe gated transport

the active transport of specific macromolecules through selective gates (such us the nuclear pore) and permits the free diffusion of small molecules b/w topologically equivalent spaces


Describe protein translocation

transmembrane protein translocators transport a protein into a across a membrane into a topologically distinct space. usually has to be unfolded. used to form integral proteins


Describe vesicular transport

membrane enclosed transport vesicles (can be small and circular or large and irregular) transport protein to a topologically equivalent compartment.


Differentiable b/w signal sequence, peptidase, and patch

a signal sequence is a stretch of 15-60 AA specifying a specific place in the cell; once sorting is complete it is cleaved by a specialized signal peptidase,
signal patches are formed by from internal signal squences that when folded create a 3D patch (used in nuclear import and vesicular transport)


What signal sequence is used to return proteins to the ER

KDEL at the C-terminus


Differentiate b/w the nuclear envelope, the inner membrane and the outer membrane

the nuclear envelope encloses DNA and defines the nuclear compartment, consists of two consecutive and continuous membranes with very different protein constituents, is penetrated by the nuclear pore. INM has binding sites of the chromosomes and for the nuclear lamina. ONM is continuous with the ER and contains ribosomes that secrete proteins into the perinuclear space.


What is the nuclear pore complex and nucleoporin?

the NPC is is a pore in the NM that acts for active selective transport and free diffusion of small molecules. Nucleoporins are the proteins that make up NPC.


What is the Nuclear Localization Sequence?

a signal sequence - consisting of one or two K and R residues, can be found anywhere on the pps, thought to form patches or loops on protein surface, only one subunit is needed in a multi complex. a nuclear import receptor is needed to bind to the NLS and transport it in. each NSR (imporins) recognizes a subset of sequences, the protein of interest can be transported folded.


What is the function of FG repeats?

They interact weakly, which gives the protein tangle gel-like properties that
impose a permeability barrier to large macromolecules, and they serve as docking
sites for nuclear import receptors


What are the different RANs

GAP converts ran-GTP to ran-GDP is on the cytosolic side while GEF on the nuclear. GAP - converts ran GDP to ran-GTP


How does nuclear import work?

receptors dock to FG repeats even with no cargo with the help of the FG repeats the receptor enters the nucleus where ran-GEF bind and causes the receptor to realize its cargo. the empty receptor with GTP goes back to the cytosol. Ran-GAP triggers Ran-GTP to hydrolyze its bound GTP, thereby converting
it to Ran-GDP


Why is there only unloading on he nuclear side?

Because the Ran-GDP in the cytosol does not bind to import (or export)
receptors, unloading occurs only on the nuclear side


How does nuclear export work?

ran-gtp bound receptor on the nuclear side promotes binding of export cargo, it then moves through the pore, meets gap which hydrolyses GTP causing the receptor to release cargo


How can we regulate import vs export?

In high Ca2+,
the protein phosphatase calcineurin binds
to NF-AT and dephosphorylates it. The dephosphorylation exposes nuclear import signals and the binding of calcineurin blocks a nuclear export signal.
The complex of NF-AT and calcineurin is therefore imported into the nucleus,.


How does the nuclear membrane dissolve during mitosis?

nuclear lamina is POted by Cdk and depolymerizes, NPC are as well and causing them to dissemble and relocate into the cytosol. ran-gef remains anchored to chromosome therefore, as you move further away from the chromosome the concentration of ran-gap increases.


What is TOM?

translocase of the outer membrane. required for the translocation of all proteins. initially transports into the inter membrane space. helps insert proteins in the membrane


What is TIM?

translocase of the inner membrane.
TIM 23 transfers soluble proteins and help insert proteins into the matrix and then into membrane
TIM 22 mediates the insertion of only certain membrane proteins from the matrix for metabolites


What is the SAM complex?

beta barrel specific sorting and assembly machinery. aid folding f beta barrel folding in the outer membrane


What is the OXA complex?

oxidase assembly protein. insertion of proteins made in the mitochondria and some that were translocated from cytosol to the inner membrane.


In what state are Mitochondria protein precursors in for translocation?

they are unfolded and stablized by chaperone proteins like HSP 70 that bind directly onto the signal sequence. pon binding to tom they are stripped from the chaperone and are inserted signal first into the organelle.


how can you determine if the protein crosses both mitochondria membranes at once or one at a time?

by cooling a cell-free mitochondrial import
system to arrest the proteins at an intermediate step in the translocation process. arrested proteins no longer contain their N-terminal signal
sequence, indicating that the N-terminus must be in the matrix space


What is mtHsp 70?

The mitochondrial hsp70 is part of a multisubunit protein assembly that is bound
to the matrix side of the TIM23 complex and acts as a motor to pull the precursor protein into the matrix space., mtHsp 70 binds to the pp being imported, conformational change. and releases the protein chain in an ATP-dependent step, exerting a ratcheting/pulling force on the protein being imported.


where does translocation to the mitochondria get energy?

outside the mitochondria ATP is needed for the pp to bind to TOM.
once bound to tim further translocation through the TIM translocation channel
requires the membrane potential made by the H+ gradient from the (H+ rich) inner membrane to the matix and one in the matrix space where mtHsp 70 uses it to act as a motor to pull the pp in.


Explain pathway 1 for translation of an interregnal protein into the inter membrane space/ IMM

usually only the N terminal SS enters the matrix, following it the stop transfer sequence (a stretch of hydrophobic AA) stops translation into the matrix. translation my tom continues and the ss is cleaved in the matrix. the stop transfer sequ is realesed my tim 23 into the membrane


Explain pathway 2for the translation of an interregnal protein into the inter membrane space/ IMM

tim 23 translates the entire protein to the matrix. a signal peptidase cleaves exposing the stop transfer sequ that directs it to the OXA complex that inserts it into the membrane.


Explain pathway 3 for the translation of an interregnal protein into the inter membrane space/ IMM?

multipass proteins dont have a N-termi ss instead they and an internal one. they completly cross with tom and then chaperones guild it to tim 22. which inserts it into the membrane. tim 22 requires ia membrane potential.


How is REDOX chem used to drive protein import?

proteins with cys motifs for disulphide bonds with Mia 40 which releases the protein in their oxidized form. Mia is in reduced form only to be deoxidized my passing e- from the ETC


What is the import signal for most peroxysome proteins?

SKL at the c-termini


Differentiate b/w co-translational and post-translational import.

co-translational import is when the ribosome is attached to the ER, as one side of the pp elongates the other side in translocated into the lumen.
post-translational- occurs for mitochondria, and chloroplast, where is synthesis is completed and the unfolded peptide is maintained for translocation.


what is the signal hypothesis?

When a ribosome translates an mRNA in vivo in the absence of the microsomes the protein synthesized was slightly longer than when translated in the presence of ER microsomes. this difference is due to the initial presence of the N-termi leader sequence that directs the secreted protein to the ER that is later cleaved by a signal peptidase before the pp is complete.


how is the ER ss directed to the ER?

a signal-recognition particle (SRP), which cycles between the ER membrane
and the cytosol and binds to the signal sequence, and an SRP receptor in
the ER membrane.


How does the SRP bind to the pp and the ribosome?

one side binds to the leader sequence while the other binds to the elongation factor binding site of the ribosome, this causes a stall that gives the ribosome time to bind to the ER membrane ensuring the pp enters the ER, this also prevents misfolding, and reduces the need for chaperones.


How is the SRP receptor involved?

once the ss and the SRP bind a receptor on the SRP for the SRP receptor is exposed. The binding of the SRP to its receptor brings the SRP–ribosome complex to an unoccupied protein translocator. the srp and the srp receptors are released and the translocator finishes the transfer.


Differentiate b/w membrane bound and free ribosomes.

they are functionally and anatomically identical only differing in the proteins that they are translating. membrane bound are attached to the cytsolic surface of the rER and translated proteins with the ER ss. while free ribosomes are used to translate all other proteins


what is sec61?

is a protein translocator complex that forms an aqueous pore across the membrane. has alpha helices that surround a central pore that is gated by a short alpha helix. when closed the the pore is impermeable. the pore is also able to open on its side allowing access to lateral transition in the hydrophobic core of the membrane.


how is post-translational import used to import to the ER?

similar to import for the mitochondria. sec 61 assessory proteins span the lumenal domain and allow HSP 70 like chaperones to bind on to the growing pp as it emerges in the ER lumen. proteins that use this route are made in the cytosol and maintain the unfolded state by chaperones.


how is the ER signal used twice?

first SRP binds to the ss. the ss also binds to specific place in the translocator, serving as a start transfer sequence opening the pore.


how are single pass proteins inserted to the membrane?

the N-termi ss starts translocation but an additional hydrophobic segment in the polypeptide chain stops the transfer process before the entire polypeptide chain is translocated.
This stop-transfer signal anchors the protein in the membrane after the
ER signal sequence has been cleaved off. with he (+) charged stretch following the internal sequ to be on the cytosolic side


How are multi-pass proteins made?

a second start-transfer sequence reinitiates
translocation further down the polypeptide chain until the next stop-transfer sequence causes polypeptides release, and so on for subsequent start-transfer and stop-transfer sequences nearly all multipass proteins lack a cleavable ss


How does SRP recognize a stretch to be a start or stop sequence?

By recognizing the
first appropriate hydrophobic segment to emerge from the ribosome, the SRP
sets the “reading frame” for membrane integration: after the SRP initiates translocation,
the translocator recognizes the next appropriate hydrophobic segment
in the direction of transfer as a stop-transfer sequence


how does glycosolation occur?

a precursor oligrosaachride is added en bloc to proteins in the ER, the sugar is added to the NH2 group of ASN by oligrosaaccharyl transferase. A special lipid molecule called dolichol anchors the precursor oligosaccharide in the ER membrane


are there any patterns to N linked glycosolation?



How does diversity in N linked glycsolation arise?

from the later modification of the original precursor oligosaccharide.
While still in the ER, three glucoses and one mannose are
quickly removed from the oligosaccharides of most glycoproteins.


what are calnexin and calreticulin?

These chaperones are carbohydrate-binding
proteins, or lectins, which bind to oligosaccharides on incompletely folded proteins
and retain them in the ER. also promote the association of incompletely folded proteins with another ER chaperone and aggregation of unfolded proteins. calanexin is in the membrane and calreticulin is soluble


How, then, do calnexin and calreticulin distinguish properly folded from incompletely folded proteins?

glucosyl transferase that keeps adding a glucose to those oligosaccharides that have
lost their last glucose. It adds the glucose, however, only to oligosaccharides that
are attached to unfolded proteins. Thus, an unfolded protein undergoes continuous
cycles of glucose trimming and addition until properly folded


Describe the process of retrotranslocation?

fails are exported from the ER, degraded by proteasome, requires energy, chaperones, and PDI, E3 ubiquitin ligase


how are missfolded proteins found?

N-linked oligosaccharides, which serve as timers that measure how long a protein has spent in the ER. The slow trimming of a particular mannose on the core oligosaccharide tree by an enzyme in the ER
creates a new oligosaccharide structure that ER-lumenal lectins of the retrotranslocation
apparatus recognize. Proteins that fold and exit from the ER faster than the mannosidase can remove its target mannose therefore escape degradation.


what is the UPR-unfolded protein response?

includes an increased transcription of genes encoding proteins involved in retrotranslocation and protein degradation in the cytosol, ER chaperones, and many other proteins to increase the folding capacity.


describe the IRE 1 path of activation the UPR

The oligomerization and autophosphorylation of IRE1 causes it to excise introns to produce an active transcription regulatory protein. This protein activates the transcription
of genes encoding the proteins that help mediate the unfolded protein response


describe the PERK path of activation the UPR

activates a second transmembrane kinase in the ER, PERK, that phosphorylates a translation initiating factor that reduces the production of new proteins. some proteins are translated when initiating factors are scare, these proteins help in activating the UPR.


describe the ATF 6 pathway

when missfolded proteins accumulate ATF6 ( a transmembrane protein) is sent to the golgi where it is cleaved by a protease. it is now able to relocate to the nucleus where it can activate genes encoding proteins for the UPR.


what is Bpi?

a resident of the ER where it binds to the lumenal tails of ATF6, PERK, and IRE 1 to supress activity, when levels of unfolded proteins increase Bip is titrated away.


how does glycosolation act as a protein anchor to membranes?

the covalent attachment of glycosylphosphatidylinositol
(GPI) anchor to the C-terminus of some membrane proteins. This linkage forms in the lumen of the ER, where, at the same
time, the transmembrane segment of the protein is cleaved off.


how does the Er assemble most lipid bilayers?

occurs exclusively in the cytosolic leaflet. fatty acid bound proteins in the cytosol help move them to the membrane. After arrival in the ER membrane and activation with CoA, acyl transferases successively add two fatty acids to glycerol phosphate to produce phosphatidic acid (water insoluble and remains in the leaflet). the polar heads are then modified giving them their chemical nature.


what is dark field microscopy?

exploits that light rays can be scattered in any direction by allowing the light to enter the sample for the side. only some of the light enters the objective lens. this creates a bright image on a black background .


what are phase contrast microscopy and differential interference contrast microscopy?

a wave's path length is changed as it refract through an organelle, the phases is shifted relative to light that has passed through an adjacent thinner. by increasing
these phase differences so that the waves are more nearly out of phase, producing
amplitude differences when the sets of waves recombine,


what is the significance of phase-contrast, differential-interference-contrast, and dark-field microscopy?

they make it possible to watch the movements involved in such processes as mitosis
and cell migration.


how does electronic image processing improve image quality?

by using CCD and CMOS, which are greatly more sensitive to light than the human eye. these cameras produce electronic images, they can be processed in various ways to extract latent information and adjust for multiple flaws.


what are the steps in sample preparation?

fix, embed, and section with a microtome.


how can we reveal the chemical make up of sells and organelles?

staining with organic dyes that have an affinity for specific sub cellular components; stains absorb light of certain wavelengths and introduce contrast by reducing amplitude
florescent probes and in-situ hybridization.


how are antibodies used in microscopy?

When labeled
with fluorescent dyes, antibodies are invaluable for locating specific molecules
in cells by fluorescence microscopy (Figure 9–16); labeled with electron-dense
particles such as colloidal gold spheres, they are used for similar purposes in the
electron microscope


how can blurs be removed in optical microscopy?

by focusing on a chosen plane in a thick specimen while
rejecting the light that comes from out-of-focus regions above and below that
deconvolution: automated image capture system with precise z stack axis control to produce a z stack that are process by the computer to deblur
confocal: only light from a point of focus is captured, decreasing detector pinhole size results in thinner optical slice.


What is FRET, how is it used and why is it important?

florescence resonance energy transfer. two molecules of interest are labels with flurophores where the emission spectrum of one overlaps with excitation of another. if the molecules are in close proximity there is a transfer of energy from one flurophore to the other, the emission of the second is seen although the molecule is excited by a photon specific to the excitation of the first.


what is photo-activation?

synthesizing an inactive form of the fluorescent molecule of interest, introducing it into the cell, and then activating
it suddenly at a chosen site in the cell; A microscope can be used to focus a strong pulse of light from a laser on a region.
Since only the photoactivated proteins are fluorescent within the cell, the trafficking, turnover, and
degradative pathways of proteins can bemonitored.


what is FRAP?

fluorescence return after photobleaching. strong focused
beam of light from a laser to extinguish the GFP fluorescence in a specified region; after which one can analyze the way in which remaining unbleached
fluorescent protein molecules move into the bleached area


explain examples of fluorescence indicators

Ca+ shifts the excitation and emissions spectra depending on their binding state
pH have spectra dependent on pH


explain single particle reconstruction

averaging method where several images of a particle are combined into one image, about 0.5nm allows to see some secondary structures


what is cryo-electron microscopy?

by flash freezing we prevent the formation of ice crystals the interrupt the structures.


how is membrane fluidity dependent on the PL?

if short and with double bonds, there is reduced interactions and less force keeping them together; reducing the phase transition point.


how is cholesterol used to maintain membrane fluidity?

Under normal conditions cholesterol enhances the permeability barrier and strengthens. b/c it prevents the hydrophobic tails from interacting, preventing phase transition.


what are lipid rafts?

specific lipids and membrane proteins seam to associate in small micro-domains = lipid rafts
help keep proteins together to function in concert,


describe the asymmetry of the plasma membrane

phosphatidylcholine and sphingomyelin are found on the outer leaflet
phosphatidylserine and phosphatidylethanoloamine are found in the inner leaflet
PS serves as an apoptosis signal


How does protein interaction limit lateral movement of lipids?

self-assembly into large aggregates
tethering to macromolecules outside the cell
tethering to macromolecules inside the cell
through interactions with proteins on an adjacent cell


how can FRAP be used to study the membrane?

by marking a membrane protein of interest with a fluoroprobe or with GFT, by bleaching an area and timing how long it takes for fluorescence to return.