Midterm 2 Flashcards
Are GLUT uniporters reversible?
Yes
What is faster, protein mediated transport or simple diffusion
protein mediated transport
What is transport rate limited by
number of transporters (Vmax) and transporter affinity (Km) for the ion
How many conformational states to uniporters have
Two
What do ATP Powered pumps do
use energy from hydrolysis of the terminal phosphoanhydride bond of ATP
What are the 4 classes of ATP powered pumps
P-class, V-class, F-class and ABC
What are P-class pumps
contain 2 identical catalytic alpha subunits that each have an ATP binding site, also contain 2 smaller beta subunits with regulatory functions. Often involved in generating ion gradients across membranes
What are V class pumps
function to generate the low pH of plant vacuoles and of lysosomes and other acidic vesicles by pumping protons from cytosolic to exoplasmic face of membrane against concentration gradient
What are F class pumps
are reverse proton pumps, also called ATP synthase
T or F: V and F class pumps have a similar structure
T
What are ABC Superfamily pumps
pump a variety of substrates, ex.multidrug resistance proteins
4 core domains: 2 T domains, form passageway for cargo. 2 ATP binding A domains, bind and hydrolyze ATP. 4 independant proteins in bacteria
what are some characteristics of the cytosol
have a pH of around 7.2, have a high K+, low NA+ and Ca2+
protein synthesis requires a high concentration of __ ions
K+
release of __ ions causes muscle relaxation, and pumped by what
Ca2+, pumped by P-class ATPase
What are some characteristics of P-class Ca2+ ATPase
2 binding sites of Ca2+, E1 states have binding sites facing CYTOSOL, E2 binding sites facing EXOPLASMIC face
E1 has __ affinity state, __ oxygen atons surround Ca2+ ions in 2 high affinity states
high, 7
E2 state has __ affinity state, has different orientations and releases __ ions
low, Ca2+
How can ATPase be converted from E1 to E2
by movements of spinning alpha helices
P class pumps are phosphorylated on an ____ residue
Asp
If Km ___ than free concentration, ions will bind
less than
__ Na+ out, __ K+ in
3, 2
Which class ATPases usually only pump H+ ions
V and F class
How do V class H+ ATPases generate electric potential
H+ ions are transported in, counter ion (OH-/Cl-) moves left of the cytoplasmic face to create an electric potential. As charge builds, the harder it is to pump H+
How do cells address positive charge build up
Let in equal number of anions, or move an equal number of different cation out of the lumen
What are some ABC Protein Flippases
ABCB1- multidrug export
ABCB4- flips phosphatidylcholine from cytosolic to exoplasmic leaflet
Is ABC CTFR a pump?
No, it is a Cl- channel
T or F: there is an electric potential across the membrane in all cells
T
What is the average charge of the membrane
-60 -> -70mV
Transmembrane potential depends on ?
ion movement
In reality, membranes are permeable to what ions?
Na, K , Ca and Cl
Animal resting membrane potential depends largely on outward flow of __ ions
K+
what are nongated channels
opening and closing of chaneels not afected by membrane potential or signaling molecules
how is the K+ gradient maintained
by the Na/K ATPase
bacterial, plant and fungal cells are inside- ______ (positive or negative) due to pumping __ out of the cell and leaving behind ____
negative, H+, anions
structure and chemical nature of a pore _____ (lowers or raises) the activation energy
lowers
what are the two forces that constitue an electrochemical gradient across a membrane
electric potential and ion concentration gradient
what type of transport is cotransport
secondary active transport
what are cotransporters
use energy released by ion movement down tis electrochemical gradient to power transport of another molecule or different ion up its concentration gradient
why in Na+ a good ion to power the uphill movement of other molecules
Na gradient is established by Na/K ATPase, Na reentry down concentration gradient movement goverened by sun of ion concentration gradient and membrane electric potential
What is the role of GLUT
transports glucose down its concentration gradient
when [glucose] in cytosol is greater than in extracellular lumen, how does glucose move
up concentration gradient via active transport, facilitated transport when opposite
what are the three antiporters that regulate the cytosolic pH and when are they active
Na+HCO3-/ Cl- , active at low pH only
Na+/H+, active at low pH only
Cl-/HCO3- , active at high pH only
synthesis of secreted proteins begins with ____ _____
cytosolic ribosomes
what sequence targets proteins to the ER
N-terminal signal sequence
what are the two key components of targeting a ribsome
signal recognition particle (SRP) and its receptor
what is the role of SRP
a cytolsoic ribonucleoprotein that binds to ER signal sequence forming a large complex. it targets nascant protein ribosome complex to ER by binding to receptor
what does the mammalian translocon consist of
3 proteins,and Sec61 complex
what is the hydrophobic hourglass
an hourglass shaped channel that has a helical plug that seals the translocon
what is signal peptidase
transmembrane ER protein that recognizes a sequence of the C-terminal end of the hydrophobic core of the signal peptide and cleaves the chain specifically
In most eukaryotes, most secreatory proteins enter the ER by ________ ________
cotranslational translocation
in yeast, how does post-translational translation occur
no SRP or receptor, Sec63 in ER near translocon. molecular chaperone BiP has peptide binding domain and ATPase domain
T or F: Intergral membrane contain one or more hydrophobic membrane-spanning domains
T
Where and how are periphrial proteins bound
to the side of the membrane by interactions with anchored membrane proteins
can membrane proteins flip flop across the membrane?
No
what is the proteins topology
the spatial orientation of proteins
how can membrane protein topology be determined
by computer programs that identify hydrophobic topogenic segments within the primary amino acid sequence
what are the 5 topological classes of intergral membrane proteins
Type I, II, III, IV, and tail-anchored proteins
which type of intergral membrane proteins have N-terminus of EXOPLASMIC and C-terminus on CYTOPLASMIC FACE
Type I and Type III
which type of intergral membrane proteins have N-terminus of CYTOPLASMIC face and C-terminus on EXOPLASMIC
Type II
how are tail-anchored proteins inserted
post-translational instertion
What are some characteristics of Type I membrane proteins
N-terminal signal sequence bound by SRP
stop-transfer anchor squence anchroed in phosphoipid bilayer
what are some characteristics of Type II and III membrane proteins
Internal signal-anchor equence bound by SRP, positive charged region stays on cytoplasmic face
Tail-anchored proteins have a __ - terminal hydroophobic tail
C-terminal
Type IV-A proteins have N terminus in the ______, while Type IV-B have N terminus in the _______.
cyotsol, ER-lumen
what are GPI anchored proteins
protein portion is synthesized and inserted to the ER membrane, like Type I protein
What is GPI transamidase
cleaves the precursor protein within the exoplasmic facing domain, covalentaly linked new C-terminus
What are the four main modifications in the ER
- Covalent addition and processing of carbohydrates (glycosylation) in the ER and Golgi
- Formation of disulfide bonds in ER
- Proper folding of polypeptide chains in ER
- Specific proteolytic cleavages in the ER, Golgi and secretory vesicles
What is the role of oligosaccharide attachment
required for some protein folding, confer stability in some glycoprotiens, play a role in cell to cell adhesion
what do disulfide bonds to to many proteins
help stabilize the tertirary and quaternary structure of many proteins, form oxidative linkage of sulfhydryl groups
what does the prescence of unfolded proteins in the rough ER do
increases transcription of genes that encode ER chaperones
Modificationso f N-linked oligosaccharides are used to monitor what?
folding and quality control
what proteins are targeted for transport from ER back to cytosol
dislocation-misfolded proteins
What is the role of the ERAD complex
enables dislocation of misfolded proteins through the membrane
what ensures proper forlding or translport in the ER
ER chaperones
Proteins require oligosaccharides and disulfide bonds via ?
ER enzymes
What class ATPase used to synthesize ATP?
F-class
Proteins on mitochrondrial and chloroplast are synthesized in ______
ribosomes
how many targeting sequences and translocation systems are required for protein import
two of each
where are precursor proteins synthesized
cytosolic ribosomes
What are the three energy imports required for protein import to mitochondrial matrix
- Cytosolic Hsp70
- Matrix Hsp70 achored to Tim44 protein
- H+ electrochemical gradient
many mitochondrial outer membrane proteins have a __ -barrel structure
beta
Antiparallel strands for _______ transmembrane segments surrounding a central channel
hydrophobic
How are chloroplast stromal proteins imported
rubisco is encoded by chloroplast DNA and transported to chloroplasts from the cytosol. stromal Hsp70 chaperone required to hydrolyze ATP
what are thykaloids
membranous sacks that contain enzymes for photosynthesis
what are the four pathways for transporting proteins from thykaloid to stroma
SecA-like
Oxa1-like
SRP-dependant
pH-dependant
what are some similarities between the SRP and pH depenadant pathways
First imported in an unfolded state, N-terminal cleaved off
What happens in SRP-dependant pathway
Unfolded protein translocated into thykaloid lumen, chaperones keep protein unfolded, thylakoid targeting sequence removed, then protein folds in thylakoid lumen
What happnens in pH- dependant pathway
folded protein transfered to thylakoid lumen, has two Arg residues, powered electrochemical gradient across thylakoid membrane, thylakoid targeting sequence cleaved off
how are targeting sequences directed in post translational transport
through translocons
what are peroxisomes
small organelles in plants and animals that contain enzymes that oxidize amino and fatty aids, breaking them down for biosynthetic pathways
do peroxisomes contain DNA and ribosomes?
No
How are Peroxisomal proteins targeted
requires C-terminal PTS1 sequence, binds to Rex5 receptor and imports folded proteins
Are peroxisomal membrane proteins targeted by PTS1
no
what are luminal peroxisomal proteins
synthesized on cytosolic ribosomes, contain c-terminal targeting sequence
what is the role of nuclear pores
allow movement of mRNAs, tRNAs, and ribosomal subunits out of the nucleus
what does the import of nuclear proteins require
nuclear localization signals
What is a nuclear pore complex
many copies of 30 distinct nucleoporins, contains nuclear basket, ions can diffuse passively but not large proteins
what are the three types of nucleoporins
Structural, membrane and F-G nucleoporins
What are structural nucleoporins
form scaffold of nuclear pore, 16 copies of Y complex
What are membrane nuceleoporins
form outer curvature of the nuclear pore
what are F-G nucleoporins
rich in hydrophobic Phe and Gly residues
How are nuclear proteins targeted
by Nuclear localization signal (NLS) nad also requires Ran and Nuclear transport receptor (Binds to NLS)
nuclear import of proteins- what happens in the cytoplasm
importin binds NLS of a cargo protein to form importin-cargo complex and diffuses thorugh NPC by interacting wtih FG nucleoporin
nuclear import of proteins- what happens in the nucleoplasm
Ran-GDP activated by GF. Releases GDP, binds GTP.
Ran-GTP bidning to importin conformational change that relases NLS-cargo protein
nuclear import of proteins- what happens in system recycling
Importin-Ran-GTP complex transported back to cytoplasm with filaments of the NPC stimulates Ran hydrolysis of bound GTP
How are proteins larger than 40kDa transported into/out of the nucleus?
Unidirectional transport though large complex nuclear pore that requires nuclear localization or export signal
What is the seceretory pathway of protein sorting
distribution of solubule and membrane protein synthesized by rough ER to final destinations at cell surface
what is the endocytic pathway of protein sorting
vesicles budding from plasma membrane take up soluble extracellular proteins and deliver them to lysosomes via endosomes
what are some techniques for studying the secretory pathway
SDS-PAGE, temperature sensitive sec mutants, cell-fre assays, can be visualized by flurescence microscopy porducing GFP tagged protein
What is the Bradford Assay
to determine protein concentration in biological samples
what are some pros and cons of the bradford assay
adv - accurate and simple, widely used in all biological fields
disadv. - requires irreversable dye binding, linear over a short range, inhibited by some detergents (SDS) , modestly time consuming
what is a good model organism for vesicular transport
yeast, as secretory pathways are similar in all eukaryotes
How does vesicle budding occur
initiated by recuritment of GTP-binding
Driven by polymerization of soluble protein
Intergral membrane domains interact with vesicle coat and cargo proteins
coat proteins shed after vesicle released
how does vesicle targeting occur
by V and t-SNAREs
Wht are V-SNAREs
incorporated into vesicle membrane during assembly of coat. are accessible after release
What are t-SNAREs
embedded in target membrane and can interact with conjugate v-SNAREs
T or F: all transport vesicles use t and v-SNAREs to fuse
T
How des vesicle fusion occur
provided by interactions of specific v-SNAREs with specific target t-SNAREs, fusion brings bilayers together and releases cargo proteins
What are Rab proteins
small GTP-bidning proteins that regulate vesicle trafficking and fusion
what are the two steps in Rab protein association with a vesicle
1- cytosolic Rab-GDP rageted to vesicle membrane-held in place by anchor
2. GEF in membrane converts Rab-GDP to Rab-GTP, which is enabled to bind Rab effectors. GTP hydrolysis releases Rab GDP
what must first be removed after it buds off parent membrane in order to expose Rab + v-SNAREs
the vesicle coat
how are SNAREs inserted into membranes
via C-terminal transmembrane chains
complexes of v and t-SNAREs are capable of reversable, tight ___-helix bundles
four
how are SNARE complexes stabilized
by arrangement of hydrophobic and charged amino acid residues in heptad residues
What are the three types of coated vesicles
COP II, COP I and Clathrin
What do COPII vesicles do
transport proteins form ER to Golgi
What do COP I vesicles do
transport proteins from Golgi eisternae and cis-Golgi back to ER
what do Clathrin coated vesicles do
transport proteins from plasma membrane and trans-Golgi to late endosomes
What vesicles contain ARF protein
COPI and Clathrin
What vesicles contain Sar1 protein
COPII
ARF, SAR1, Ran and Rab are all ______ switch proteins
GTPase
What is the other function of small GTPases
pinching off completed vesicle from parent membrane
what do luminal domains do that are on some cargo proteins
identify and bring soluble cargo proteins into the vesicle
Newly synthesized proteins travel in ____- vesicles from ER to the __-Golgi
COPII, Cis-golgi
retrograde transport recycles v-SNAREs in ____ vesciles from cis-Golgi to __
COPI, ER
Yeast cells with mutations in genes for COPII proteins are class __ sec mutants
B
Sar1-GTP binds to ER memnrane and recruits ____ and _____
Sec23 and Sec24
Cytosolic domains of COPII contain____
di-acidic sorting signal
Certain intergral ER membrane proteins also bind cargo proteins in the lumen via _____ ____
exoplasmic domain
All signal domains interact with ____ in COPII
Sec24
How do vesicles move?
Many move by diffusion in the cytoplasm, microtubules act as railroad tracks to transport vesicles
what is retrograde transport
COPI vesicles transport between Gogli cisternae and from cis-golgi to rough ER
What are some characteristics of ER-resident proteins
many soluble proteins that assist in folding and modification of newly synthesized proteins, most carry KDEL sorting sequence
what is the role of KDEL receptor
acts mainly to retrieve soluble proteins containing KDEL sorting signal at end of C-terminal segment
what is cisternal maturation
responsbile for anterograde transport thorough the Golgi (cis to trans)
What fusion can demonstrate cisternal maturation
Fluorescence-tagged fusion
