Midterm 2 Flashcards
1
Q
Ran is a ______ switch protein
A
GTPase
2
Q
describe the secretory pathway of protein sorting
A
distribution of soluble and membrane proteins synthesized by the rough ER to final destinations at the cell surface
3
Q
describe the endocytic pathway of protein sorting
A
vessicles budding from plasma membrane take up soluble extracellular proteins and deliver them to lysosomes via late endosomes
4
Q
what are two techniques used for studying the secretory pathway?
A
sds page, analysis of yeast temperature sensitive secretory mutants, cell free asssays
5
Q
protein transport through the secretory pathway can be visualized by____________ microscopy of cells producing a GFP tagged membrane protein
A
fluorescence
6
Q
transport of a membrane glycoprotein from the ER to the golgi can be assayed based on sensitivity to cleavage by ___________
A
endoglycosidase D
7
Q
what is a bradford assay used for ?
A
determine the protein concentration in biological samples
8
Q
what are two advantages and two disadvantages of bradford assay?
A
accurate, widely used, simple, alternates are more expensive, requires irreversible dye binding, linear and short range, inhibited by detergents, time consuming
9
Q
why are yeasts a good model organism for vesicular transport?
A
secretory pathway and molecular components similar in all eukaryotes
sec mutants in yeast were identified to secrete proteins at one temp but unable at higherq
10
Q
the golgi apparatus outputs to the ______ ______ and inputs to the ____
A
plasma membrane, ER
11
Q
how does vessicle budding work?
A
bud from the parent donor organelle and fuse with particular target organelle
12
Q
what is budding initiated by?
A
recruitment of gtp binding proteins to a patch of donor membrane
13
Q
how is vessicle budding driven?
A
by polymerization of soluble protein complexes on the cytosolic face of the membrane
14
Q
after budding when the vessicle is released ____ proteins are shed
A
coat proteins
15
Q
what are v snares?
A
v for vessicle, incorporated into vessicle membrane during assembly of coat, initially occluded by vessicle coat
16
Q
t snares are?
A
for target, are embedded in target membrane and can join with cognate v snares
interaction brings membrane close together
17
Q
all transport vessicles use v snares and t snares to fuse (t or f)
A
true
18
Q
what are rab proteins?
A
small gtp binding proteins that regulate vesicle trafficking and fusion with the approprite membrane
19
Q
_____ anchor allows rab proteins to be tethered to the vesicle membrane
A
isoprenoid
20
Q
what is the two step process of the association of a Rab protein with a vessicle?
A
- cytosolic Rab GDP is targeted to vesicle membrane and held in place by isoprenoid anchor
- GEF in the membrane converts Rab GDP to Rab GTP which is enabled to bind Rab effectors
21
Q
after vessicle fusion ______ hydrolysis releases Rab GDP
A
GTP hydrolysis
22
Q
in order to expose both Rab and v snares, what must occur?
A
vessicle coat must be removed after it buds off parent membrane
23
Q
rab mediated docking does what?
A
to cognate Rab effector occurs first
24
Q
interaction of _______and ________ close enough to fuse
A
v snares and t snares
25
snares are ?
abundant proteins that are usually inserted into mebranes via c terminal transmembrane domain
26
cytosolic region of each snare protein contains what?
repeated heptad sequence of 60-70 amino acids that confer ability to form coiled coil structures
27
what are the 3 types of coated vesicles?
COPII - vesicles trnasport proteins from the ER to the golgi
COPI - vesicles mainly transport proteins in the retrogade direction between golgi cisternae and from the cis golgi back to the ER
Clathrin - coated vesicles transport proteins from the plasma membrane and the trans golgi network to late endosomes
28
what is the GTP binding protein that controls coat assembly in COPI and clathrin coated vessicles?
ARF protein
29
what is the GTP binding protein that controls coat assembly in COPII vessicles?
Sar1 protein
30
what are the 4 steps to vessicle coat assembly?
1. sar 1 membrane binding gtp exchange
2. COPII coat assembly
3. GTP hydrolysis
4. coat assembly
31
what are the 4 switch proteins?
ARF Sar1 Ran and Rab
32
how does the vesicle coat selects cargo proteins by?
directly binding specific sorting signals of cytosolic domains of transmembrane cargo proteins
33
what exposes Rab and SNARE proteins that target vessicles for fusion with specefic target mebranes?
coat shedding
34
how is the first step of the secretory pathway mediated?
by COPII vesicles
- contain v snares
- move in anterograde direction
35
v snare recycling and retrieval of missorted ER resident proteins from the cis golgi is via COPI coated vesicles in the _______ direction
retrograde
36
newly synthesized proteins travel in COPII vesicles from the ER to the cis golgi in which direction?
anteriograde
37
retrograde transport recycles what?
v snares in COPI vesicles form cis golgi to ER
38
yeast cells with mutations in genes for COPII proteins are class ___ sec mutants and accumulate proteins in the _____ ____.
B, rough ER
39
how is formation of the COPII vesicles triggered?
sec12 catylyzes activation of cytosolic Sar1
40
Sar1 GTP binds to ER membrane and recruits _____ and _____
sec 23, sec24
41
how does sec24 interact with the cytosolic domain of COPII ternary complex?
with di acidic targeting signals that recruits cargo protein into the vesicle membrane
42
the cytosolic domains of COPII contain what?
di acidic sorting signals, the signal binds to sec24 subunit of COPII coat
43
all sorting signals interact with Sec24 (t or f)
true
44
how do vessicles move?
by diffusion in the cytoplasm
45
how is the ER to golgi intermediate compartment formed?
several ER derived vesicles fused (aka cis golgi network)
46
retrograde tansport is?
between golgi cisternae and from cis golgi to rough ER
47
What proteins assist in folding and modification of newly synthesized secretory proteins?
chaperone BiP, protein disulfide isomerase, many soluble proteins
48
most ER resident proteins carry what?
a KDEL sorting sequence at the c terminus
49
what is the function of the KDEL receptor?
acts mainly to retrieve soluble proteins containing the KDEL sorting signal
50
KDEL receptor contains ___________ at the end of the c terminal segment
KKXX sorting signal
51
_________ _____ can also target certain proteins to COPI vesicles
Di arginine sequence
52
what is cisternal maturation responsible for ?
anterograde transport through the golgi
53
vesicle based transport visible by electron microscopy are all _____ transporters
COPI
54
as retrograde transport occurs the enzymatic makeup of the _______ _______ changes
cisternal lumen
55
how do COPII coated vesicles transport newly synthesized proteins containing golgi targeting sequences?
in their cytosolic domain or bound to such proteins from the rough er to the cis golgi
56
how do COPI coated vesicles transport vesicles carrying ER/golgi resident proteins?
in the retrograde direction which supports golgi cisternal maturation
57
facilitated transport is movement ____ the concentration gradient?
down
58
secondary active transport and primary active transport is movement _____ the concentration gradient?
up
59
p class Na /K ATPase moves ___ Na ions out of the cell and ____ K ions into the cell
3, 2
60
what is the E1 conformation of p class na/k atpase?
three high Na binding sites and two low affinity K binding sites accessible from the cytosolic surface of the protein
61
what is the E2 conformation ?
the three bound Na ions gain access to the expolasmic face and simultaneously, the affinity of the 3 Na binding sites drop
62
open/closing not affected by membrane potential or signaling molecules so called ____ ___________
non gated channels
63
simple diffusion rate of substance movement across the phospholipid part of a membrane is proportioal to its ______ ______, ______ and _______
concentration gradient, hydrophobicity and size
64
type 1 single pass transmembrane protein have N terminus on ______ face, and the C terminus on _______ face
exoplasmic, cytoplasmic
65
how is the signal anchor sequence bound in type III single pass transmembrane proteins?
SRP binds the terminal singal anchor sequence synthesized by a cytosolic and interacts with SRP receptor on the ER membrane ribosome
66
where are precursor proteins synthesized?
cytosolic ribosomes
67
polypeptide can be kept unfolded by ?
chaperones, cytosolic Hsp70
68
during protein import into the mitochondrial matrix ____ is inserted into the translocon of the outer membrane?
MTS
69
MTS is removed by a matrix ______
protease
70
a pure lipid bi layer is impermeable to ?
ions, amino acids, sugars, most water soluble molecules
71
gases and small uncharged water soluble molecules use _______ as transport
diffusion
72
would life be possible for a pure lipid bilayer without proteins?
no
73
the higher the k value the more _________ and faster to cross a bilayer
hydrophobic
74
all membrane transport proteins are ______ integral membrane proteins
multipass
75
channels transport water, ions and hydrophobic small molecules ____ concentration gradient
down
76
_____ is faster and more specific than ________
uniport, diffusion
77
in uniport the molecules dont enter the bilayer directly ( t or f)
true
78
the lower the km the more _____ the substrate binds to the transporter
tightly
79
which transport is the fastest?
| mannose or glucose
glucose transport 3.8 times faster
80
galactose is the slowest transport (t or f)
true
81
what is osmotic pressure?
the hydrostatic pressure required to stop the movement of water caused by concentration gradient
82
aquaporins _____ water permeability of cellular membranes?
increase
83
what type of transport is faster than simple diffusion?
protein mediated transport
84
________ proteins convert between two conformational states to facilitate diffusion across a membrane
uniport
85
what is f class ATPase used for ?
to synthesize ATP
86
what does indebendent DNA encode?
rRNA trnas
87
most proteins are encoded by?
nuclear genes
88
N terminal targeting sequences specify binding to receptor proteins on the ______ organelle?
surface
89
protein import requires ______
energy
90
where does double membrane transport occur?
outer and inner membranes are in direct contact
91
double membrane transport requires what?
two targeting sequences and two translocation systems
92
where are matrix targetting seuqences are located?
at the N terminus
93
how long are matrix targetting sequences ?
20 to 50 amino acids
94
does transport to the mitochondria involve the ER or the golgi complex?
no
95
what are the 3 energy inputs required for import?
cytosolic Hsp70, matrix has Hsp70 anchored to the Tim44 protein, H+ electrochemical gradient across inner membrane
96
proteins must be ________ to translocate
unfolded
97
what is the path A for mitochondrial intermembrane space?
analogous to type one membrane, protein are inserted into the ER
98
many mitochondrial outer membrane proteins have ______ structure
b barrel
99
what is tom44?
general import pore
100
what is the SAM complex?
sorting and assembly machinery comlex, inserts b barrel proteins into the outer membrane
101
the chloroplast lumen is called?
stroma
102
enzymes of the calvin cycle fix ______
co2
103
rubisco is encoded by?
chloroplast dna
104
small subunit of rubisco and other calvin cycle enzymes contain _______ _______ sequence
N terminal stromal import sequece
105
chlorplast stroma sequence is located at the _______
N terminus
106
endoplasmic reticulum sequence is located at the ________
N terminus
107
Mitochondrion sequence is located at the _______
N terminus
108
Peroxisome sequence is loctaed at the?
C terminus for most
109
import of chloroplast stromal proteins uses post _________ ______
translational translocation
110
the stromal Hsp70 Chaperone is required for ?
import of proteins into the mitochondrial matrix
111
Hsp 70 does what ?
hydrolyzes ATP
112
Thylakoids are?
internal interconnected membranous sacs that contain all the enzymes for photosynthesis
113
many thylakoid proteins are synthezised in the ?
cytosol
114
what are the 4 pathways for transporting proteins from the stroma to the thylakoid?
1. Sec1 like
2. Oxa1 like
3. SRP dependent
4. pH dependent
115
descripe the srp dependent pathway? **
- used for transport of plastocyanin and related proteins
- first imported in unfolded state into chloroplast stroma
- n terminal stromal sequence cleaved off
- unfolded protein translocated into thylakoid lumen
- chaperones keep unfolded
- thylakoid targetting seuqence removed then protein folds in thylakoid lumen
116
descripe the ph dependent pathway? **
- used by proteins that bind metal containing cofactors
- first imported in unfolded state into chloroplast stroma
- n terminal stromal sequence cleaved off
- folded protein translocated into thylakoid lumen
- Tat pathway
- thylakoid targetting sequence must have two closely spaced arg residues
- tat proteins oligomerize in membrane to create pores of variable size to accomidate folded protein
- powered by electrochemical gradient across thylakoid membrane
- thylakoid targetting sequence cleaved off
117
mitochondrial and chloroplasts proteins encoded by nuclear genes are synthesized and maintained in an unfolded state by _______ this is _________ ________
chaperones, post translational translocation
118
what do targetting sequences direct?
post translational transport of the unfolded proteins through translocons into the organelles
119
name two characteristics of peroxisomes?
small organelles, bound by single membrane, divide when large enough, in mammals not abundant in liver, dont have dna and ribosomes
120
what is the function of peroxisomes?
contain enzymes that oxidize amino acids and fatty acids breaking them down for biosynthetic pathways,
121
All luminal proteins must be transported into _______
peroxisomes
122
targetting of peroxisomal proteins requires a ________ _______ ________ sequence?
c terminal ser lys leu targetting sequence, called peroxisomal targetting sequence 1 (PSI1)
123
PTS1 binds to ______
Pex5
124
Peroxisome import machinery imports ______ proteins
folded
125
ubiquitinylation controls ____ recycling back into monomeric soluble form in cytosol
Pex5
126
peroxisomal membrane proteins are not targetted by PTS1 sequences ( t or f)
true
127
luminal peroxisomal proteins are synthesized on free cytosolic ribosomes and contain a ______ targetting sequence
c terminal
128
the nucleus is separated from the cytoplasm by what two membranes?
nuclear envolope and continuous with er
129
what are nuclear pores used for?
allow movement of mrnas trnas, ribosomal subunits out of the nucleus
130
import of nuclear proteins requires ?
nuclear localization signals
131
structural nucleoporins form?
scaffold of nuclear pore including 16 copies of the y complex
132
what are the 3 nucleoporins?
structural nucleoporins
membrane nucleoporins
FG nucleoporins
133
what is the nuclear localization signal (NLS) ?
directs selective transport into the nucleus
134
targetting of nuclear proteins requires?
Ran and nuclear transport receptor
135
what ensures the direction of nuclear import ?
localization of GEF in the nucleus and GAP in cytoplasm
136
do aquaporins have conformational change?
no
137
what are the 4 classes of atp powered pumps?
P Class
V class
Fclass
ABC superfamily
138
describe p class
combined action of p class atp powered pumps generates usual ionic milieu of animal cells
139
v class and f class transport what?
most transport H+
140
abc superfamily transports?
transports substrates, drugs, toxins, peptides, phospholipids
141
name two characteristics of P class pumps
two catalytic a subunits, smaller beta subunits, generate ions across membranes
142
describe V and F class pumps?
v class - function to generate low ph of plant vacuoles and of lysosomes and other acidic vesicles in animal cells by pumping protons from the cytoplasmic to exolasmic face
f class- reverse proton pumps also called atp synthases
143
describe atp superfamily pumps
atp binding casssete superfamily
| most diverse class, multidrug resistance proteins
144
what are the 4 core domains of abc superfamily ?
two transmembrane t domains, two cytosolic atp binding domains bind and hydrolyze atp
145
muscle relaxation requires?
Ca2+ ATPases
146
what is stored in the sarcoplasmic reticulum?
ca 2+ ions are concentrated and stored here
147
_______ ca atpase located in the SR membrane pumps ca from the cytosol back into the lumen of the SR
p class
148
what makes up more than 80 % of intergral membrane protein in muscle sr membranes
p class ca 2+ atpase
149
what is the difference between the e1 and e2 states of pclass ca2+ atpase?
e1 - binding site facing the cytosol
e2 - have binding sites facing the exoplasmic face of the membrane
150
what has a higher affinity, the e1 or e2 state?
e1 high affinity
| e2 low affinity
151
where are p class pumps phosphorylated?
on an Asp residue
152
why are v class H+ atpases called electrogenic pumps?
because a net movement of electric charge occurs during transport
153
what are two ABC proteins ?
ABCB1 multidrug export
| ABCB4 flips phosphatidylcholine from cytosolic to exoplasmic leaflet
154
what is ABC CFTR?
cl channel
has a and t domains
cl flows down concentration gradient
155
resting membrane potential results from?
charge separation across the membrane, -70mv cytosolic face negative with respect to exoplasmic face
156
nongated ion channel transports Na+ and K+ Ca 2+ and Cl - ________ concentration gradient
down
157
the animal cell plasma membrane is generated by ?
ATP powered na/k pump and nongated K channels
158
what two forces constitute an electrochemical gradient across a membrane?
electric potential and ion concentration gradient
159
what is the Na/ glucose symporter?
GLUT transport glucose down concentration gradient, facilitated transport, cyclic mechanism
160
what is symport mechanism best studied for?
bacterial Na/ amino aicd symport
161
_____ ion bound to carboxyl group of transported leucine in the symport mechanism
Na+
162
polarized cells are ________
asymetric
163
what is the two stage movement of molecules through a cell?
import through apical membrane
| export though the basolateral membrane
164
what are three transport processes involved with apical and basolateral plasma membrane regions of epithelial cells transport proteins?
transport of amino acids and glucose from intestinal lumen to the blood
stomach acid secretion
bone resorption
165
what begins on cytosolic ribosomes?
synthesis of secreted proteins, integral plasma membrane proteins, proteins destined for lysosomes, golgi, er or plasma membrane
166
membrane bound ribosomes are recruited to the ER during synthesis of a polypeptide containing an ________
er signal sequence
167
when cells are homogenized the plasma membrane shears the rough er into small vessicles called __________
microsomes
168
what is cotranslational insertion?
cell free expierments demonstrate that translocation of secretory proteins into microsomes is coupled to translation
169
what are two key componants of targetting ribosomes to the RER or SRP?
signal recognition particle and its receptor
