Midterm 3 Flashcards
1
Q
what are the components of the endomembrane system
A
- rough ER
- Smooth ER
-Endosomes
-lysosomes
-transition /transport/secretory vesicles - perioxisomes
- vacuoles
1
Q
what are the RER and SER the sites for
A
lipid and protein synthesis
2
Q
what do lysosomes do
A
digest ingested material and unneeded cellular components
3
Q
what does a peroxisome do
A
house hydrogen peroxide generating reactions
4
Q
what do vacuoles do
A
store ions, sugars, amino acids, and toxic compounds
5
Q
what do endosomes do
A
transport
6
Q
what is the lumen
A
the internal space in the ER
7
Q
where are polypeptides synthesized
A
- 1/3 in the RER
- 2/3 on free ribosomes
8
Q
what polypeptides are synthesized in RER
A
secreted proteins
transmembrane proteins
soluble proteins that reside in ER, Golgi, lysosomes, edosomes, vesicles, vacuoles
9
Q
what polypeptides are synthesized in free ribosomes
A
proteins destined to remain in cytosol
peripheral proteins of the cytosolic surface on membranes
proteins that are transported to the nucleus
proteins that are incorporated into peroxisomes, chloroplasts, and mitochondria
10
Q
where do all proteins begin synthesis
A
on ribosomes in the cytosol
11
Q
how does ER know where proteins go
A
info is coded in ER signal sequence
12
Q
where does translation start
and what happens
A
the cytosol
mRNA leaves nucleus and associates with free ribosomes
13
Q
when do polypeptides diverge to what pathway for routing and what are they
A
after translation
free ribosomes
ER docked ribsomoes
14
Q
what are the 2 pathways protein sorting involves
explain them
A
- co-translational import : proteins carrying an ER sequence direct the ribosome polypeptide complex to RER, translation is completed on RER
- post translational import: proteins lacking an ER signal sequence complete their synthesis on free ribosomes , and are then released into cytoplasm with organelle-specific sorting signal
15
Q
what sorting proteins do cytoplasmic proteins have
A
none
16
Q
what is a polysome
A
multiple ribosomes synthesizing from the same mRNA
17
Q
what does the signal hypothesis propose
A
that intrinsic molecular signals determine the location of some polypeptides
18
Q
what happens if a sorting signal is deleted
A
targeting of protein is lost
19
Q
what does the ER signal sequence usually have
A
hydrophobic N-terminal region and a polar region near the cleavage from the mature protein will take place
20
Q
what does the signal recognition particle (SRP) bind
A
binds to ribosome mRNA polypeptide complex to the ER membrane
21
Q
what does the SRP contain
A
proteins and RNA
22
Q
what happens when the SRP binds to the signal sequence
A
blocks further translation
23
Q
what happens once the SRP has been released
A
the ER signal sequence is inserted into the translocon (a channel protein)
this contact displaces the plug, opening the channel to the ER lumen
As protein elongates it passes into the lumen
24
what happens when polypeptide synthesis is complete (RER)
the polypeptide is released into the lumen and the ribosome detaches from the ER membrane
subunits dissociate and release mRNA
25
what do GTPases (G proteins) do
act as molecular switches
26
what is an example of a G protein
SRP
27
what are the two types of membrane proteins
single and multi-pass
28
what does asymmetry reflect
function
29
where are oligosaccharides always present
on the non-cytosolic side
30
where can hydrophobic transmembrane domains dissolve into lipid bilayer
through a seam along one side of the translocon
31
what determines the orientation of a multipass/integral protein
determined by the charge/orientation of the first transmembrane domain
32
what must each subsequent transmembrane domain have
opposite charge as one before
33
what is the ER the site of
protein synthesis
protein modification/maturation
recognition and removal of misfolded proteins
lipid synthesis
34
how does ER modify/mature proteins
glycosylation ( = glycoproteins)
folding of polypeptide chains and subunit assembly
disulphide bond formation
35
how does ER recognize and remove misfolded proteins
ER associated degradation (ERAD) recognizes misfolded proteins
36
where do misfolded proteins get degraded
cytosolic proteases
37
what happens to most proteins are the ER
they are glycosylated
(N-linked glycosoylation)
38
what does Golgi do in glycoslyation
modifies it
adds O linked sugars
39
what do carbohydrate groups function as
macromolecule bonding sites
aid in protein folding
increase stability
40
when is the oligosaccharide added to the recipient protein
and what is it called
as the polypeptide is being synthesized
called co-translational glycosylation
41
where do polypeptides fold into their final shape (ER)
in ER lumen
42
what are lectins and give 2 examples
carbohydrate binding proteins
Calnexin and calreticulin
43
what do calnexin and calreticulin do
bind to N linked oligosaccharides, preventing aggregation and promoting proper folding
drives disulphide bond formation
44
when does calnexin and calreticulin binding happen
after processing the oligoscacchardide precursor has begun
since they recognize a single terminal glucose
45
what is binding protein (BIP)
a chaperone in ER lumen
46
what does BIP do
binds to hydrophobic regions of polypeptide chains and prevents aggregation of polypeptides with similar regions
prevents interaction between hydrophobic regions of different proteins
47
what does the enzyme protein disulphide isomerase (PDI) do
catalyzes disulphide bond formation in ER lumen
48
in eukaryotic cells where is the only place disulphide bonds are formed
ER lumen
49
where do disulphide bonds form when protein is being synthesized
between adjacent cysteines
50
what activates the unfolded protein response (UPR)
when proteins are made faster than they are folded, processed, and transported
51
what happens when UPR is activated
- phosphorylate translation factors (inhibits protein synthesis to decrease the flow of proteins into ER)
- upregulate the expression of: ER based chaperones, transport that move proteins out of ER, and protein degradation machinery
52
where are degraded proteins identified by ERAD degraded
in proteasome in cytosol
53
what are proteasomes
large protein degrading structures
54
what do proteasomes bind to and what do they do after
ubiquitin-labelled proteins
they hydrolyze peptide bonds in an ATP-dependant manner
55
what joins ubiquitin to target protein
by a process involving 3 enzymes:
- ubiquitin activity enzyme (E1)
- ubiquitin conjugating enzyme (E2)
- ubiquitin ligase or substrate recognition protein (E3)
56
what is the primary source of membrane lipids including phospholipids and cholesterol
ER
57
where are fatty acids for membrane phospholipids synthesized
cytoplasm
58
how do fatty acids get from cytoplasm to lumen side of bilayer
transferred by phospholipid translocators (flippases)
59
what does the the type of phospholipid transferred across the membrane depend on and what does it lead to
the particular translocator present
leads to membrane asymmetry
60
what of lipid lipid transfer proteins do and what does this allow
exchanges lipids between compartments
allows for the movement of phospholipids to membranes outside the endomembrane system
61
62
what does the golgi complex do
further processes and sorts glycoproteins and membrane lipids playing a central role in membrane and protein trafficking in eukaryotic cells
63
where are proteins released into ER lumen routed to
golgi, secretory vesicles, lysosomes, or back to ER
64
what is the TGN and what does it do
Vesicle sorting station
segregating proteins into vesicles headed to plasma membrane or other intracellular destinations
65
what is the CGN and what does it do
transition vesicle sorting system
sorting of proteins to be sent back to the ER or onto the golgi
66
what is glycosylation
addition of carbohydrates side chains to proteins
67
what is terminal glycosylation and where does it occur
modifications of glycoproteins through the removal/addition of sugar side chains on the core oligosaccharide (which is added in ER)
This occurs in golgi
68
what side of membrane does glycosylation occur
luminal (interior)
69
what is each step of glycosylation strictly dependent on
the preceding modification
addition of next sugar relies on the presence of the previous carbohydrate
70
what are the functions of glycosylation
-participate in protein/lipid sorting in the trans-golgi network
- makes glycoproteins/membranes more resistant to digestion by proteases by creating the glycocalyx
- serve as recognition molecules in cell-cell interaction
- regulatory roles (Poetin folding/stability), blood type, immune regulation
essentially gives cell ability to generate many chemically distinct molecules on cell surface
71
what are the two hypotheses for movement through golgi
stationary cisternae model: cisternae and the resident enzymes stay in place, while cargo moves from one stack to the next
cisternae maturation model: cargo remain within a cisternae, while the cisternae move forward ( cis to trans) and resident enzymes shuttle backwards in vesicles
72
what is anterograde transport and example
movement of material toward plasma membrane
ex: exocytosis
73
what is exocytosis
fusion of vesicle with the plasma membrane, releasing their contents into the extracellular
74
what is retrograde transport and an example
movement of materials towards ER and away from plasma membrane
ex: endocytosis
75
what is endocytosis
formation of vesicle on the plasma membranes, taking up solutes from the extracellular space
76
what are the ways that a protein is kept within an organelle
- retention: resident molecules are excluded from transport vesicles
- retrieval: tags can be used to return escaped proteins to their proper locations
77
what is a tag made of
depending on the protein/destination can be an amino acid sequence, a hydrophobic domain, or oligosaccharide side chain, or something else
78
what is the most common ER retrieval tag
KDEL retrieval signal
returns soluble protein back to ER
79
what is the sequence in KDEL
Lys-Asp-Glu-Leu
80
what is the most common amino acid sequence for ER specific retrieval/retention signals
Lys-Lys-any-any
81
what happens if KDEL proteins leave ER (they are ER residents)
they associate with KDEL receptor
binding is efficient in the lower pH environment of the golgi
82
what do KDEL receptors mediate
packaging of KDEL proteins into ER bound transport vesicles
83
when KDEL receptors release KDEL proteins in ER is the binding efficient
no, since ER has high pH
84
what are the functions of the proteins vesicles are coated in
- curve the membrane to form the vesicle
- select the components to be carried by the vesicle
85
what are the 2 protein layers vesicles are composed of and what do they do
outer layer: acts as scaffolding to cage around the vesicle
inner layer: acts as an adapter between the outer layer and the lipid bilayer/cage. Selects cargo proteins through affinity interactions with cytoplasm domains or these transmembrane proteins
86
what are the classes of coated vesicles
COP11 coated vesicles
COP1 coated vesicles
clathrin coated vesicles
87
what do COP11-coated vesicles do
move materials from the ER forward (integrate transport) to the ERGIC (ER-golgi intermediate compartment, AKA vesicular tubular cluster) and golgi
88
what do COP1- coated vesicles do
move materials from the ERGIC and golgi backward (retrograde transport) or from the trans golgi to the cis golgi cisternae
89
what do the clathrin-coated vesicles do
move materials from the plasma membrane backwards and the TGN to the endoscopes/lysosomes/plant vacuoles
90
does COP11 or COP1 associated with budding
COP11
91
what initiates budding
by recruitment of the small GTP binding protein (GTPases) SAR-GDP to a patch of donor membrane
92
how do vesicles move to their destination
mediated by microtubules
like a train (motor protein) pulling cargo (vesicle) along train tracks (microtubules)
93
what does the initial contact between a vesicle and a target membrane involve
tethering proteins
94
what are the 2 types of tethering proteins
rod shaped fibrous proteins that form long bridges
multi protein complexes that hold 2 membranes close together
95
what is most of the specificity in tethering proteins conferred by
Rab GTPases
96
how does Ran GTPase work
it receipts the cytosolic tether proteins to the membrane surface, allowing docking to occur
also plays a role in recruiting motor proteins to vesicles to facilitate transport
97
what do SNARE proteins do
mediate fusion between vesicles and target membranes
98
what does the SNARE hypothesis explain
states that the sorting and targeting of vesicles involves 2 families of SNARE (snap receptor) proteins
99
what are the 2 families of SNARE proteins
V SNARE: vesicles SNAP receptor. found on vesicles
T SNARE: target SNAP receptors, found on target membranes
100
How to V and T SNARES pull membranes together
alpha helices intertwine
101
what does dissociation of V and T SNARES require
NSF (N-ethylmalemide-sensitive factor) and SNAPs (soluble NSF attachment proteins) to pry apart the SNARES using energy from ATP hydrolysis
102
what happens after V and T SNARES are separated
V SNAREs are shuttled back to the previous compartment to repeat the process
103
what is constitutive secretion
after budding form TGN, vesicles move directly to the cell surface and immediately fuse with the plasma membrane
104
what is regulated secretion
secretory vesicles that accumulate in the cell and only fuse with plasma membrane in response to specific signals (ex: Ca 2+)
105
what is polarized secretion
specific proteins are secreted form a limited region of the plasma membrane
106
give 2 examples of cels that use polarized secretion
neurotransmitters are only secreted at nerve junctions
intestinal cells secrete digestive enzymes out of the intestinal side of the cell
107
how does polarized secretion work
proteins/lipids destined for polarized secretion are sorted into vesicles that have receptors that bind to localized sites on plasma membrane
108
what is a primary way cells maintain their membrane fluid balance
bulk phase endocytosis (clathrin - independent endocytosis)
109
is bulk phase endocytosis specific
no
110
what rate does bulk phase endocytosis go at
constant
111
what does bulk phase endocytosis compensate for
plasma membrane gains by exocytosis and maintaining surface to volume ratio
112
how can checks selectively and efficiently acquire macromolecules
by receptor mediated endocytosis (clathrin dependent endocytosis(
113
what are the steps of receptor mediated endocytosis
1. specificity molecules (ligands) bid to their receptor on the outer cell surface
2. as the receptor-ligand complex diffuse laterally they encounter specialized regions called coated pits (sites for collection and internalization of these
3. accumulation of complexe sin the pits trigger the accumulation of clathrin-coat proteins on the cytosolic side of the membrane inducing a curvature of the pit
4. pit will eventually pinch off, forming a coated vesicle s
114
do clathrin coated vesicles differ from COP11 vesicles in their formation and proteins
yes
115
what is required as clathrin accumulates around the budding vesicle and what does it do
dynamin
constricts and closes the vesicle
116
is dynamin a GTPase
yes
117
what is phagocytosis
ingestion of macromolecules, cell parts, or whole organisms
118
in humans what cell does most of phagocytosis
white blood cell
119
how does phagocytosis work
contact with the target triggers the onset of phagocytosis, as folds in the membrane (pseudopods) surround the object forming an intracellular phagocytic vacuole (or phagosome)
120
what happens to mist material taken up by cell
destroyed by lysosomes
121
what acid do lysosomes contain
acid hydrolyses capable of degrading major classes of macromolecules
122
what is acid hydrolase
a hydrolytic enzyme with optimal pH of 5
123
are lysosome lumen's basic or acidic
and why
very acidic
essential for the functioning of acid hydrolases
124
what is a endocytic vesicle
the vesicle budding from the plasma membrane as a result of bulk phase or receptor-mediated endocytosis
125
what is an early endosome
the primary sorting station in the endocytic pathway
126
what is a late endosome
an organelle containing the full complement of acid hydrolase but who's lumen has not reached the pH of 4-5
127
what is a lysosome
an organelle that is digestively active (all hydrolases and correct pH)
128
What are the 2 routes for a late endosome to mature into a lysosome
1. the ATPase pumps can lower the pH of the late endosome, which activates the enzymes
2. the late endosome can fuse with an existing lysosome
129
what are the 2 types of receptors in endocytic pathway
housekeeping receptors
signalling receptors
130
what does a housekeeping receptor do
mediate the uptake of material that will be used by the cell
131
how does signalling receptor work
bind extracellular messengers that change the activity of the cell
132
what happens after signalling receptors bind
receptors get degraded (receptor down regulation), reducing the sensitivity of the cell to the further simulation
133
what is a residual body
indigestible materials that remain in the lysosome
134
what do cells do with cellular waste
some release contents
some accumulate them and it contributes to cellular aging
135
what is autophagy
the digestion of old organelles
136
what is macrophagy
formation of an autophagic vacuole (autophagosome) when an organelle becomes wrapped in a double membrane derived from the ER
137
what is macrophagy
a small vacuole is formed surrounded by a single membrane
138
what allows polypeptides to enter organelles after they've been synthesized
post-translational import
139
what do proteins getting imported to the ER need
- sorting signal
- receptor
- membrane transporter
140
what is a peroxisome
(structure and defining characteristic)
spherical organelle, bounded by single membrane
defining characteristic is the presence of catalase
141
what are the functions of peroxisomes
- H2O2 metabolism (balance)
- detoxification of harmful compounds
- oxidation of fatty acids
- metabolism of N-containing compounds
- catabolism of unusual substances
142
how are peroxisomes formed
generated by division of peroxisomes or by vesicle fusion
143
how do peroxisomal proteins get into peroxisomes
either synthesized by peroxisomal enzymes
or transported there by lipid transfer proteins
144
where are most peroxisomal enzymes synthesized and transported
free ribosomes
post translationally imported via a C terminal peroxisomal targeting signals (PTS 1)
145
where are most mitochondrial and chloroplast polypeptides synthesized
free ribosomes
146
what allow passage of small molecules in outer membrane in mitochondria/chloroplasts
porins
147
how does translocation across both membranes work in mitochondria
occurs simultaneously at sites where the inner and outer membranes are close together
uses transport complexes called TOM and TIM
148
what does TOM and TIM stand for
Translocase of the Outer Mitochondrial membrane
Tranlocase of the Inner Mitochondrial membrane
149
how is targeting of mitochondrial proteins done
1 - cytosolic proteins are bound by molecular chaperones to keep them in an unfolded state
2- presequences encoded by am N-terminal alpha helix (containing positively charged and hydrophobic amino acid residues) binds the receptor component of TOM, and is positioned into the TOM
3- during transport of proteins destined for the matrix, the TOM and TIM channels are brought together
4- movement into matrix is powered by electrical potential ( matrix is more negative, pre sequence is more positive)
5- then mitochondrial proteins are pulled into matrix ( either by chaperones or Radom diffusion)
6- once in the matrix, the presequence is cleaved by a peptidase and the protein is folded into its native conformation
150
what do all chloroplast proteins have
a stroma targeting domain which is removed by a peptidase upon transport
151
what is a thylakoid transfer protein needed for
for entry into the thylakoid
(for nuclear genes)
152
are some thylakoid membrane proteins co-translationally inserted
yes, chloroplast genes
153
through what does traffic continuously travel through between nucleus and cytosol
nuclear pores
153
what are nuclear pores
specialized channels in the nuclear envelope where inner and outer membranes are fused
provides direct contact between cytosol and nucleoplasm
154
what speed to small particles pass through nuclear pores
speed proportional to their size
155
how are proteins that are too big to freely diffuse getting in nucleus
through active transport
nuclear localization signals (NLS) and nuclear export signals (NES) enable proteins to be recognized and transported by the nuclear pore complex
156
what is the general structure of NES and NLS
contain proline and positively charged amino acids
157
what provides energy to transport proteins through nuclear pores
hydrolysis of GTP by GTPase
158
what does protein isolation start with
mechanical disruption (homogenization)
chemical disruption (lysis)
159
what is homogenization
rupturing cell to release the cell contents
160
what do we want cell homogenates to have
- disrupted plamsa membrane
- released soluble proteins
- maintain the integrity of organelles (or create microsomes)
161
why do you often add protease inhibitor in lysis
to prevent protein degradation
162
what is an isotonic buffer used for in lysis
break apart cell but not the organelles
163
what gives you a cell slurry
combination of mechanical and chemical disruptions
164
can harsh lysis buffers (amphipathic detergents) be used
yes, though these denature the protein
165
what separates different organelles/micromolecules in centrifuges
different sedimentation rates
166
what is differential centrifugation
separation of particles/organelles of different sizes and density by their rate of sedimentation
167
in differential centrifugation what size particles end up on bottom
larger ones
(bigger sedimentation coefficient)
168
what is sedimentation coefficiemt relative to
size/density of a molecule
169
what does sedimentation coefficient measure
how rapidly the particle sediments when subjected to centrifugation
170
how does density gradient centrifugation work
samples are placed on top of gradient (often sucrose) and centrifugation causes particles to move into discrete zones based on their density and the density gradient
dense particles move into gradient faster
171
what is chromatography
a procedure where a mixture of dissolved components is fractionated as it moved through some type of porous matrix (usually a column)
172
what does ion-exchange chromatography rely on
differences on surface charge of macromolecule
173
how does ion exchange chromatography work
beads carry a weak charge, and charged proteins react to beads to varying degrees, separating them
174
what happens as pH is lowered in ion exchange chromatography
negatively charged amino acids become neutral
positively charged amino acids become more abundant
175
what does gel filtration chromatography rely on
differences in size and shape of macromolecule
176
how does gel filtration chromatography work
beads have different sized pores that allow proteins to go through them gradually
larger proteins get through faster
177
what does affinity chromatography rely on
specific interactions between a ligand/substrate/antibody and the target
178
how does affinity chromatography work
beads have specific molecules attached to their surface and preferentially bind to the protein being purified
179
what is immuniorecipitation
when antibodies are used in affinity chromatography
180
what type of chromatography usually results in a near total purification of the desired molecules in a single step
affinity chromatography
181
what is electrophoresis
a group of techniques that use an electric field
182
what is electrophoresis useful for
for characterizing a mixture of soluble proteins
183
what does how quickly a molecule moves during electrophoresis depend on
shape, charge, size
184
what are detergents
amphipathic molecules that disrupt the lipid bilayer, disrupts hydrophone interactions, and thus dissolved the lipids
185
what often happens prior to electrophoresis
insoluble and soluble proteins are exposed to high heat and SDS which disrupts protein-protein and protein-lipid associations
186
what are examples of disulphide bond reducing agents
Beta - mercaptoethanol
dithiothreitol (DTT)
187
how does an SDS PAGE work
- denature sample with SDS
- Apple mixture of proteins on gel, apply electric field
- negatively charged proteins/SDS move towards positive Botton of gel
- smaller ones move faster
- proteins end up in decreasing size down the gel
188
what is 2D SDS PAGE
separates proteins in 2 dimensions
first by charge, then by size
189
how does mass spectrometry work
1. proteins are digested by a protease to break them into smaller polypeptides
2. polypeptides are separated by their mass/charge ratio
3. computer analysis compares the spectrum with a database
190
what is western blotting (immunoblotting)
after electrophoresis (SDS PAGE), polypeptides can be identified using it
proteins are transferred to a membrane and bound by specific antibodies
191
what do hydridomas do
fuse a lymphocyte (antibody producing cell) to an immortalized myeloma cell (able to divide forever)
can produce a single antibody type and secrete it in the culture media for collection
192
what are the steps of western blotting (immunoblotting)
1. mixture is separated by SDS PAGE
2. transferred to a membrane using an electric current
3. incubated with a primary antibody, wash, then secondary antibody
4. react with substrate and image
193
what is a genome
entire set of info on DNA (includes coding and non coding)
194
wha is a transcriptome
entire complement of RNA expressed by a genome
195
what is a proteome
entire complement of proteins expressed by genome in a cell
196
what is an interactome
map of the network of interactions between proteins in an organisms proteome
197
what can hub proteins have
several binding sites or can bind different proteins at same time
single binding interface which is capable of binding several proteins at different times
198
are hub proteins more or less likely to be essential for cellular survival
more likely
199
what is chromatin immunoprecipitation (CHIP) for
finds where a protein binds within the genome of a cell
200
what are 3 techniques to determine the 3D structure of a protein
X ray crystallography
nuclear magnetic resonance spectroscopy
cryo-electron microscopy
