Cell Bio Exam 3 Flashcards
1
Q
What are the 2 main parts of the vesicular transport?
A
Vesicle budding and fusion
2
Q
Default Pathway Route
A
Goes from RER to Golgi to plasma membrane (PM)
3
Q
2 signals required to divert from the default pathway
A
- Retention signals
- diversion signals
4
Q
Function of Golgi Apparatus
A
Plays critical role in biosynthesis, sorting, dispatching, and recycling of proteins to various parts of the cell
5
Q
Structure of the Golgi apparatus
A
From entrance to exit:
1. cis golgi network
2. cis cisterna
3. medial cisterna
4. trans cisterna
5. trans golgi network
6
Q
Function of the perinuclear (aka golgi apparatus)
A
To remove and add sugars to glycoproteins through the action of resident glycosidases and glycotransferases
7
Q
What molecules in the golgi add on sugars
A
glycosyltransferases
8
Q
What molecules in the golgi cleave sugars?
A
glycosidases
9
Q
What 2 parts of the golgi apparatus make up the “sorting reticulum”
A
- cis golgi network
- trans-golgi network
10
Q
Purpose of cis-golgi network
A
passes proteins to the cis-golgi stack or returns them to the ER
- basically sorts what comes into the golgi
11
Q
Purpose of trans-golgi network
A
passes proteins to the plasma membrane, lysosomes, or secretory vesicles
- sorts where proteins go after exiting the golgi
12
Q
purpose of the medial golgi
A
this is where the glycosidase and glycosyltransferase reactions occur
13
Q
Purpose of the intra-golgi
A
Transport vesicles
14
Q
What is the rate-limiting step in protein transport and secretion?
A
Export from the ER
15
Q
Questions that ER machinery asks
A
Is the protein correctly folded and assembled with other subunits
16
Q
If ER detects that proteins are not correctly folded what occurs?
A
The denatured or unfolded protein is degraded
17
Q
What occurs if the ER detects that the protein is not correctly assembled with other subunits?
A
If protein is still attached to BiP or other chaperones, and is actively still undergoing folding, then proteins remains in the ER
18
Q
What is BiP
A
BiP is a heat shock protein (part of HSP70 family) found in the ER that helps protein with folding
19
Q
What occurs if the ER detects that the protein is correctly folded and assembled with other subunits?
A
The protein will be exported by the default pathway to the cis-Golgi network
20
Q
What occurs to misfolded p in the ER lumen?
A
A misfolded p will be moved out back through the Sec61 complex where it will bind to a N-glycanase. This will lead to ubiquitin binding to it and the p will be degraded by a proteosome
21
Q
2 ER Retention Signals
A
- BiP
- PDI
22
Q
What is the sequence for ER retention signals?
A
KDEL, short AA sequence
23
Q
What binds to KDEL sequence
A
KDEL receptors present in ER and cis-Gogli
24
Q
KDEL Recycling Steps
A
- Secretory proteins and ER resident protein receptors are moved along the default pathway from the ER to the golgi. ER resident p are also sent to golgi in separate vesicles
- ER resident p containing KDEL sequence bind to the ER p receptors in the golgi and are then recycled back to ER
- return requires use of Microtubules
25
Name for KDEL Retrieval Mechanism
Retrograde Transport
26
KKXX Sequence
sequence that is present in some membrane p that can be recycled back to ER
27
What happens if the KDEL Sequence is mutated
Protein will go to the cell surface and not be recycled back to ER
28
Proteins in Rough ER are either....
1. resident
2. en route to other destinations
29
What requirements must proteins meet before they can leave the RER?
1. folded
2. modified
3. assembled correctly for proper function
30
3 molecule types that ensure that a protein is ready to leave the ER
1. Chaperones
2. Isomerases
3. Glycosyltransferases/glycosidases
31
ER Chaperones
BiP (HSP 70 family member)
calnexin
calreticulin
32
Monoglucosylated glycoproteins
Calnexin and calreticulin are Ca dependent and help with p folding
33
Function of ER Chaperones
- Prevent aggregation of hydrophobic domains
and facilitate folding in ATP-dependent manner
- help retain partially folded p in ER avoiding premature transit to golgi
- also facilitate translocation (similar to mt-hsp7- activity)
34
Function of isomerases
ex: protein disulfide isomerase (PDI)
Help form disulfide bonds to prevent p from going to golgi
35
Function of glycosyltransferases and glycosidases
Glycosyltransferases (adding sugars)
Glycosidases (cleave sugars)
36
Glycosylation Function
P in ER lumen are post-translationally glycosylated
37
How are Oligosaccharide Precursors Formed?
Oligosaccharides assembled from nucleotide and lipid phosphate monosaccharides in the cytosol and ER through a sequential series of transfer reactions. Precursor is first assembled on lipid carrier called dolichol
38
Steps of Synthesis of Oligosaccharide Precursor
On cytosol side
1. phosphorylation of dolichol by ATP hydrolysis
2. 2 nucleotide UDP-GlcNAc are added to the dolichol
3. 5 GDP-Man are added to doichol complex
4. Flipping occurs in membrane
On lumen side
5. 4 dolichol phosphate Man are added to the dolichol complex
6. 3 dolichol phosphate Glc are added to dolichol complex
39
What occurs after oligosaccharide precursor is assembled?
oligosaccharide is transferred to the Asn residue of a protein in the ER lumen
40
What catalyzes the transfer of oligosaccharide precursor to a protein?
Oligosaccharyl transferase
41
What complex is associated with oligosaccharyl transferase that help speed up p modifications after translocation to lumen?
Sec61 complex
42
What is removed from the high mannose glycan complex before it exits the ER?
terminal 3-Glc and 1-Man are removed before the complex exits ER
43
What occurs to the glycan complex once it reaches the golgi?
Further removal/addition of sugars using:
- glycosidases (remove)
- glycosyltransferases (add)
44
What are the 2 types of attachment bw oligosaccharide and Asn
N-linked or O-linked
45
What is the consensus glycosylation sequence
A-X-Ser/Thr
X can be any AA except Proline
46
What are the 2 types of oligosaccharides studied in class
1. Complex oligosaccharides
2. high mannose oligosaccharides
47
What are the 3 destinations that particles are sent to after sugar processing occurs in the golgi
- lysosome
- PM
- secretory vesicle
48
What are the 2 golgi structure models
1. vesicular transport model
2. cisternal maturation model
49
What is the vesicular transport model
Model that explains that golgi is static and transport vesicles ferry cargo between stacks
50
What is the cisternal maturation model
Model that explains that as Golgi cisternae mature, they move forward through stack
- at each stage, Golgi resident proteins carried forward are moved backward via vesicular transport
51
What is endocytosis
vesicular import of particulates and macromolecules
52
What is stored in lysosomes
acid hydrolases that are used for degradation
53
What hydrolases are found in lysosomes
nucleases, proteases, glycosidases, lipases, phosphatases, sulfatases, phospholipases
54
What is required to keep the low pH in lysosomes
The H+ pump which is a V-type ATPase that acidifies lysosome environment
55
What are the 4 pathways used to deliver material to the lysosome
- biosynthetic pathway from ER-golgi
- endocytic pathway from outside environment and brought into cell by endocytosis
- autophagic pathway (self destruct)
- phagocytic pathway (endocytosis in specialized cells like macrophages and neutrophils)
56
what synthesizes and sorts lysosomal hydrolases and membrane proteins
Golgi
57
How are lysosomal hydrolases and membrane proteins delivered to lysosome
delivered via transport vesicles that traffic through late endosomes
58
What are the 2 ways lysosomal proteins are specifically selected for delivery to late endosomes/lysosomes relative to other pathways?
lysosome hydrolases have 2 types of molecular signals
- signal patch
- M6P marker
59
How does a signal patch ensure that hydrolases are delivered to lysosome
A signal patch is read by glycosyltransferases in cis-Golgi that add an M6P tag to proenzymes
60
How does a M6P tag ensure that hydrolases are sent to lysosome
M6P is read by M6P receptors present in the trans-Golgi network then M6P bind and segregate hydrolases from the other protein traffic exiting the Golgi
61
Steps of hydrolase traveling to lysosome
1. lysosomal hydrolase precursor from ER has M6P tag added in cis-golgi network
2. precursor then travels to trans network where M6P receptors recognize the tag and bind
3. Clathrin-coated transport vesicle is created and travels to late endosome (receptor dependent transport)
4. Vesicle fuses with late endosome where H+ pump triggers the release of M6P tag
5. M6P receptor is dissociated from precursor due to acidic pH of late endosome
6. M6P receptor budding vesicle is created and recycled back to trans-golgi network
62
What are LAMPs
lysosome associated membrane proteins that follow the default secretory pathway to the plasma membrane
63
What is another name for lysosome membrane protein pathway
scavenger pathway
64
What path do the majority of lysosome direct proteins take to arrive at lysosome?
ER to golgi to late endosome to lysosome
65
What is the alternative pathways for proteins to arrive at lysosome
Lysosome membrane protein pathway
66
What path do proteins take in scavenger pathway
ER to golgi to PM to early endosome to late endosome to lysosome
67
Pathway to PM in scavenger pathways is also followed by?
small percent of hydrolases and M6P receptors
68
What are 2 lysosomal storage diseases that occur due to mutations
1. hurler's disease
2. I-Cell disease
I stands for inclusion
69
What is mutated in Hurler's disease
Lysosomal ez required for breakdown of glycosaminoglycans (ECM proteins) is missing
70
What is mutated in I-cell disease?
Defect in GlcNAc phosphotransferase causing there to be no M6P tag
71
What is the effect of no M6P tag in I-Cell disease?
M6P receptors fail to sort and target hydrolases
72
Function of endocytosis
- uptake of nutrient molecules
- clearance of harmful substances, cell debris and infectious organisms
- maintenance of surface-to-volume ratio
73
3 types of endocytosis
1. phagocytosis
2. receptor mediated endocytosis
3. pinocytosis
74
What occurs to majority of ingested material during endocytosis?
material is delivered to lysosomes for degradation
75
Phagocytosis function
- cell eating
- uptake of large particulate matter
- engulfs material
76
Receptor mediated endocytosis function
uptake of macromolecules via specific cell surface receptors
- ligands are smaller in size
77
Pinocytosis function
- "cellular sipping/drinking"
- uptake of fluid and dissolved solutes
- uptake of bulk volume
78
2 main features of phagocytosis
1. practiced by a select group of cells
2. receptor-mediated event
79
In what cells does phagocytosis occur?
- protozoa (giant amoebae) ingest food particles
- macrophages and neutrophils (binding, clearance, and destruction of bacteria, damaged cells, and cellular debris)
- fibroblasts (remodeling of connective tissues)
80
How is phagocytosis a receptor-mediated event
- cells possess surface receptors that recognize specific or generic sites on various particles
- cells bind particle then progressively extend pseudopodia to engulf particles
- engulfment requires receptor-ligand interactions around surface of particle
81
How does the pseudopodia extend?
1. receptor ligand interactions induce localized changes in cortical cytoplasm
- Ca2+ influx induce formation of gel like cytoplasm
- membrane coupled pseudopod extension and actin polymerization
2. engulfment completed when pseudopodia meet and fuse forming phagosome
82
2 ways that ingested particles are degraded?
1. oxygen metabolites help kill bacteria for particles that reside in phagosomes
2. the acid hydrolases digest contents in phagolysosomes
83
What are phagolysosomes
product of fusion of lysosomes with phagosomes
84
What are residual bodies
phagolysosomes containing non-digestible material
85
3 ways that pathogens use to avoid phagocytosis degradation
1. escape
- break phagocytic walls
2. prevent fusion w/ lysosomes
- don't bind with lysosome
3. survive in phagolysosome
- survive in harsh environment w/ thick layer of pathogens
86
Example of escape phagocytosis mechanism
listeria virulence
87
Steps in Listeria virulence
1. listeria attaches to E-cadherin
2. uptake by zipper mechanism
3. hemolysin secretion
4. hemolysin mediated membrane disruption
5. bacterial release and replication
6. hemolysin destroyed in host proteosome
7. released bacteria will grow tail using actin nucleation
8. bacteria moves w/ actin tail to PM
9. Protrusion formation occurs before bacteria is engulfed by neighboring host cell
88
2 mechanisms to attach bacterium to host cell
1. zipper mechanism
2. trigger mechanism
89
Zipper mechanism steps
1. The adhesin on bacteria will bind to adhesin receptors on host cells
2. leads to actin filament increasing and absorbing bacterium
90
2 examples of adhesin receptors
integrins and cadherins
91
Trigger mechanism steps
1. type 3 secretion apparatus on bacterium binds to the host cell
2. activates the Rho-family GTPase
3. activation triggers actin polymerization
4. actin polymerization then absorb bacterium
92
Pathogen that uses integrin adhesin receptor
Yersinia uses invasin-beta1 integrin
93
Pathogen that uses e-cadherin adhesin receptor
Listeria
94
2 main features of Receptor mediated endocytosis
1. vesicular uptake of macromolecules
2. uptake mediated by specific cell surface receptors that increase efficiency of uptake
95
Macromolecules in RME
1. Nutrients
- Lipoproteins : LDL and HDL
- metals: Fe
2. Hormones
3. growth factors
4. immunoglobulins
5. harmful substances
- modified glycoconjugates or coagulation
factors
96
Cholesterol in RME
Cholesterol is take up via LDL receptors and delivered to lysosomes
97
Why are some people predisposed to heart attacks
Genetic analysis shows defective LDL receptors that result in increase in blood serum cholesterol levels
98
Why do specific cell surface receptors lead to an increase in efficiency?
- bind ligands with high affinity
- receptor ligand complexes enter cells specialized membrane structures aka clathrin coated pits
99
What are clathrin-coated pits?
Specialized endocytic structures that collect endocytic receptors
100
2 results of clathrin coated pits
1. exclude other membrane proteins
2. concentrate endocytic receptors
101
What 2 effects would occur if clathrin adaptor proteins were defective?
1. reduced binding to cargo (LDL)
2. defective cytoplasmic tails
102
what are the 2 proteins involved in coated pits?
1. clathrin
2. adaptor complexes (proteins)
103
Structure of clathrin
3 heavy chains and 3 light chains which form a heterohexamer
104
what is the building block for clathrin
a triskelia/triskelion
105
what does the structure of a clathrin coat look like
when clathrin triskelion self assemble they form a lattice composed of hexagons and pentagons like a soccer ball
106
Function of clathrin adaptors
complexes that mediate clathrin binding to specific membrane receptors
107
types of clathrin adaptors
1. plasma membrane AP-2
2. trans-golgi network AP-1
108
What is the purpose of endocytosis signal motifs?
the endocytic receptors possess 4 AA motif that mediate interaction with coated pit proteins
109
What is the key feature of an endocytosis signal motif
Protruding loop with aromatic amino acid, often Tyr
110
2 examples of endocytosis signal motifs sequences
1. NPXY (LDL receptor)
2. FRXY (transferrin receptor)
111
Clathrin Cycle steps
1. Adaptor proteins are recruited to PM
2. Triskelia bind to adaptor complex
3. Triskelia reorganize into soccer ball
4. the stalk of pit invagination is pinched off using dynamin
5. Clathrin uncoating mediated by clathrin associated hsp ATPase
112
What are the main 3 endosomal compartments
1. early endosome
2. late endosome
3. lysosome
113
What is the function of the early endosome
sorting and recycling center
114
What is the function of the late endosome?
depository for ligands targeted for degradation
115
What is the function of lysosome?
House acid hydrolases and fuse with late endosomes to then be able to degrade proteins
116
What is the pH of the different endosomal compartments?
1. early endosome
- pH = 6-6.5
2. late endosome
- pH = 5.5-6.0
3. Lysosome
- pH = <5.5
117
RME Pathway for LDL
1. endocytosis
2. uncoating
3. fusion with endosome
4. LDL trafficked to lysosome
5. LDL degraded in lysosome and release of free cholesterol
6. LDL receptor is recycled back to PM
118
What triggers the release of glucose receptors to PM
Insulin binding to insulin receptor triggers release of glu receptors to PM to boost glu uptake into the cell
119
Retrieval Pathway
When protein receptors are recycled back to the golgi complex
- ex: M6P receptor back to trans-golgi
120
Biosynthetic Pathway
LAMPs and M6P receptor positive
- when particle travels along default pathway up the PM and is then endocytosed (early to late to lysosome)
121
Endocytic Pathway
LAMP positive and M6P receptor negative
- when external components are endocytosed (PM to early to late to lysosome)
122
Types of Neiman Pick Diseases
Type A, B, and C
123
What deficiency occurs in Neiman Pick Type A and B diseases?
Ez deficiency of acid sphingomyelinase (ASM) which is ez that breaks down sphingolipids. This causes a buildup of sphingolipids in the cell causing the cell to die and damage organs
124
Features of Type A Nieman Pick Disease
Associated with neurological tissues and usually causes death within 2-3 years
125
Symptoms of Type B Neiman Pick Disease
Enlarged spleen, respiratory problems, cardiovascular problems, can live into adulthood
126
What mutation occurs in Type C Neiman Pick Disease?
Mutation in NPC1 protein which is needed for cholesterol transport. Leads to accumulation of cholesterol in late endosome and it cannot be released. Causes block of retrograde transport of M6P receptors to the golgi transport
127
Steps that lead to degradation of receptors?
1. receptors tagged with ubiquitin are endocytosed to early lysosome
2. Ubiquitin is recognized by ECRT protein complexes (1, 2, 3) leading to invagination of receptor into the late endosome
3. Multivesicular body combines with transport vesicle that contains lysosomal proteases and lipases.
4. Receptors w/ ubiquitin tags are degraded by proteases and lipases in the lysosome
128
Example of when receptor needs to be degraded
Growth factor
129
How do some MVBs undergo exocytosis
Limiting membrane of MVB fuse with PM and ILV are released converting them to exosomes
130
Steps in transcytosis
1. Antibody binds to Fc receptor on the apical membrane and is endocytosed
2. Goes to early endosome then moves to recycling endosome
3. Receptor and antibody are then transcytosed to the basal membrane
4. Recycling vesicle with PM returns to apical membrane
131
In what cells does transcytosis occur?
In polarized cells such as epithelial cells (intestine or bloodstream)
132
What are the 5 possible fates for Receptors and Ligands?
1. receptor recycled and ligand degraded
2. receptor and ligand recycled
3. receptor and ligand are degraded
4. Receptor and ligand move from one membrane domain to another (transcytosis)
5. Receptor recycled and ligand stored
133
Example of when receptor is recycled and ligand is degraded
LDL receptor and LDL particles
134
Example of when both receptor and ligand are recycled
Apotransferrin and Transferring receptor
135
Example of when receptor and ligand are degraded
Epidermal Growth factor, EGF receptor
136
Example of when receptor and ligand are moved from one membrane domain to another
maternal IgGs, when a baby receives antibodies during lactation
137
Example of when receptor is recycled and ligand is stored
vitelligenin in oocytes
138
What is pinocytosis
uptake of fluid, nonspecific volume taken up in bulk
139
Where does pinocytosis occur
in all eukaryotic cells
140
2 pathways of pinocytosis
clathrin dependent and independent pathways
141
Functions of pinocytosis
1. non-specific uptake of solutes
2. routine turnover of PM proteins and lipids
3. maintenance of surface-to-volume ratio
142
what are caveolae?
non-clathrin coated membrane structures
143
main protein in caveolae?
caveolin
144
Potential functions of caveolae
1. pinocytosis (clathrin independent)
2. uptake of small solutes
3. site of cell signaling
145
what is exocytosis?
vesicular export of molecules out of the cell
146
General features of exocytosis
- membrane p and lipids delivered to provide new components to PM while soluble cargo can be secreted to extracellular space
- 2 main pathways
147
2 pathways of exocytosis
1. constitutive transport
2. regulated secretion
148
function of exocytosis
maintain balance of inward membrane flow with outward membrane flow
149
Purpose of constitutive transport
- transports secretory vesicles from the TGN to the PM
- delivers proteoglycans and glycoproteins out of cell
150
Purpose of Regulated secretion
- special class of secretory vesicles that rely on rapid, signal dependent release
151
What is regulated pathway dependent on
assembly of vesicle is clathrin dependent, microtubule and actin cytoskeleton dependent, and release of cargo is signal dependent
152
Essential features of constitutive secretion
- flow is continuous, does not require signal
- supplies PM with new lipids and proteins
- movement is clathrin independent
- cargo is not concentrated
153
Examples of constitutive secretion
serum albumin, transferrin in mammalian hepatocytes
154
What cell types follow regulated secretion
- neurons, Beta islet cells, mast cells, goblet cells, sperm, eggs
- neurotransmitters, insulin, histamine, mucins
155
How does TGN specifically sort and concentrate molecules for regulated secretion
Use a sorting signal thought to be some type of patch that lead to distinct secretory vesicles (or granules)
156
Are regulated secreted vesicles clathrin dependent
yes, they use a clathrin mediated mechanism to bud vesicle
- involves acidification by V-type ATPase
157
What is used to move vesicles in regulated secretion pathway?
Kinesin (microtubules) and myosin 1 (actin) needed for movement and actin rearrangement helps with exocytosis release
158
what adaptor protein is used in vesicle budding in regulated secretion
Requires AP-1 for clathrin mediated budding of golgi derived vesicles
159
What occurs after budding to the secretory vesicle in exocytosis
immature secretory vesicles are uncoated and undergo further maturation which help to condense vesicle contents
160
How are proteins packaged during exocytosis when in immature secretory vesicles?
they are packaged in a precursor form and are further process prior or just after secretion
- ex: polypeptide hormones, neuropeptides, and hydrolytic enzymes
161
Examples of precursor proteins stored in immature secretory vesicles
- proinsulin to insulin
- digestive enzymes
162
what is the advantage of storing proteins in precursor form
cells will release cargo only when needed and signal is sent
163
What 2 changes occur as vesicle is matured in exocytosis
1. cargo concentration increases
2. cargo is acidified w/ V-type
164
how are vesicles apposed to the PM
enmeshed in the cortical actin-membrane cytoskeleton network
165
What is required for release of cargo once it is at the PM
A signaling event is required and trigger secretory granules to fuse with PM and release cargo to extracellular space
166
What intracellular signal occurs when specific ligands bind receptors on cell surface
Influx of Ca levels
167
Increase in intracellular Ca levels leads to
- transient disruption or fragmentation of cortical cytoskeletal network
- vectorial movement of vesicle to the PM where membrane fusion and secretion occur
168
Steps leading to exocytosis
1. vesicle arrives with help of microtubule and docks on cortex
2. ligand binds receptor which trigger rise in Ca levels, disruption in actin network, and vesicle interaction with PM
3. vesicle membrane fuses with PM releasing content of vesicle
169
When does mast cell degranulation occur
when cell is introduced to allergen that the body detects as an invader and results in granules releasing histamine
170
Can regulated exocytosis be localized
yes, an exp demonstrated that when histamine is released at localized cite, after mast cell exposure to stimulant coupled to a bead, exocytosis only occurred at site of contact with bead
171
What does allergy medication do
Allergy medicine is an antihistamine that blocks released histamine to prevent histamine from triggering allergy symptoms such as inflammation and increase in body temp
172
what does protein efflux from TGN require
both signal mediated diversion to lysosomes and signal mediated diversion to secretory vesicles require clathrin coating
173
What are the 2 routes of protein sorting from TGN in polarized cells
1. direct pathway
2. indirect pathway
174
what is the direct pathway for protein sorting from TGN in polarized cells
from TGN to correct PM domain (apical or basal)
175
What is the indirect protein sorting pathway from TGN in polarized cells
From TGN to incorrect domain
- protein then transported to correct domain via transcytosis
- ex: protein that are meant for basal membrane are sent to apical membrane by accident and are then transcytosed to basal membrane
