Endomembrane System Part 1 Flashcards
1
Q
Transport Vesicles
A
large amounts of material trafficked between each organelle/structure by these small, membrane-bound molecules
2
Q
Donor Membrane Compartment
A
cargo-containing vesicle buds off this membrane compartment
3
Q
Acceptor Membrane Compartment
A
vesicle transported to this membrane compartment
4
Q
Vesicle Coat Proteins
A
select which donor membrane and soluble lumenal cargo proteins enter the transport vesicle and regulate vesicle formation and budding
5
Q
Biosynthetic Pathway
A
materials transported from ER to golgi, endosomes and then lysosomes
6
Q
Constitutive Secretion Pathway
A
materials continually transported from golgi to plasma membrane and/or released by exocytosis outside of the cell in secretory vesicle
7
Q
Regulated Secretion Pathway
A
- only in specialized cells
- ER-derived materials from golgi stored in secretory granules
- secetory granules fuse with plasma membrane and release by exocytosis lumenal cargo into extracellular space
- secretory granule membrane components incorporated into plasma membrane
8
Q
Endocytic Pathway
A
- operates in opposite direction of secretory pathway
- materials from plasma membrane or extracellular space incorporated into cell and then transported to endosomes and lysosomes
9
Q
Pulse-Chasing Radiolabeling and Autoradiography
A
- experiments demonstrated how proteins move through the secretory pathway
- pancreatic tissue briefly incubated (pulse) with radioactive amino acids which are incorporated into newly-synthesized proteins
- tissue washed and incubated (chase) for varying lengths of time with non-radioactive amino acids
- protein synthesis continues and radiolabeled proteins traffic through cell
- tissue fixed and killed and exposed to X-ray film - autoradiography
10
Q
Live-Cell Imaging With Autofluorescent Proteins
A
- gene-encoding autofluorescent protein (GFP, RFP etc.) linked to gene-of-interest
- recombinant gene fusion introduced by cloning into selected organism/tissue/cell
- intracellular localization and trafficking of fluorescent fusion protein visualized in living specimen using fluorescence microscopy
11
Q
Subcellular Fractionation
A
isolation of organelles by centrifugation
12
Q
homogenization
A
- type of subcellular fractionation
- cell/tissue disrupted while ensuring that organelles remain intact
13
Q
homogenate
A
- result of homogenization
- gets filtered to remove unbroken cells and large fragments
- subjected to differential centrifugation
14
Q
supernatant
A
liquid at top of centrifuge tube
15
Q
differential centrifugation
A
seperates intact organelles/cellular components of different size/density with increasing higher centrifugation speeds
16
Q
Microsomes
A
fragments of ER membrane and/or plasma membrane that fuse and reform into small, spherical vesicles
17
Q
Equilibrium density-gradient centrifugation
A
- separates organelles/cellular components on basis of density
- determine composition of isolated organelles using proteo/lipodomics and/or use in cell-free import and vesicle trafficking assays
- organelle fraction layered on top of** sucrose gradient** (density increases from top to bottom)
- individual organelles migrate to corresponding equilibrium densities
- different layers of gradient removed and purified organelle fractions identified by EM and/or organelle marker proteins/enzymes
18
Q
cell-free systems
A
- characterization of the activities of specific endomembrane protein components in vitro (components purified from different organelle/ER microsomal fractions
- liposomes mixed with purified proteins
- allows for processes underlying protein/vesicle trafficking in endomembrane system to be reconstituted in vitro
19
Q
liposomes
A
- proteins are incubated with liposomes in cell-free systems
- artificial, spherical vesicles consisting of phospholipid bilayer surrounding aqueous center
20
Q
Mutant Phenotype Analyses
A
- “genetics approach”
- to identify genes/proteins and steps in protein/vesicle trafficking in endomembrane system by screening for mutant phenotypes
21
Q
Yeast sec mutants
A
- conditional mutants
- collection of temperature-sensitive mutants that secrete proteins at permissive temperature but not at higher nonpermissive temperature
- accumulate normally secreted proteins at points in endomembrane pathway blocked by mutation and/or possess defects in organelle morphology and/or distribution
- 5 classes
- double mutants indicate order of steps in pathway
22
Q
Endoplamsic Reticulum
A
- starting point for biosynthetic and secretory pathways
- site of protein and lipid synthesis, protein folding and processing/quality control
- network of membrane-enclosed, rod-like tubules and sheet-like cisternae
23
Q
Endoplasmic Reticulum lumen
A
- aqueous space inside ER tubules and cisternae
24
Q
Endoplasmic Reticulum Cisternae and Tubules
A
- shapes mediated by reticulons
- undergo bending, growth, shrinkage, fusion, fission
- make the ER highly dynamic
25
Rough ER
* subdomain of ER
* mostly cisternae with bound ribosomes
* protein and membrane phospholipid synthesis
26
Smooth ER
* subdomain of ER
* mostly curved tubules lacking ribosomes
* Ca2+ storage and hormone synthesis
27
Mitochondria-Associated-Membranes and Plasma-Associated-Membranes
* ER regions that make direct contact with mitochondria or plasma membrane
* membrane protein and lipid exchange
28
ER Exit Sites
ER regions where transport vesicles bud off enroute to Golgi
29
Free Ribosomes
* in cytoplasm
* fate of nascent properly-folded soluble or membrane protein in cytoplasm: remain in cytoplasm OR target to proper intracellular component
30
ER Membrane-Bound Ribosomes
* bound to ER
* fate of nascent properly-folded soluble or membrane protein in RER: remains in ER, localizes to other ER subdomain, localizes to other ER-derived organelles, targets from ER onto another compartment in endomembrane system
31
Co-Translational Translocation
* protein targeting to and across ER membrane
* uses signal sequence, SRP, SRP receptor, Sec61 translocon, a-helix plug, signal peptidase
32
Signal Sequence
* in Co-translational translocation:
* a stretch of 8-15 hydrophobic amino acids on the N-terminus of the nascent growing polypeptide
33
Signal Recognition Particle
* In co-translational translocation
* recognizes exposed signal sequence on polypeptide
* binds to ribosome and stops protein translation
34
SRP Receptor
* in co-translational translocation
* SRP complex binds to it
* GTP hydrolysis releases the complex
35
Sec61 translocon
* simultaneously with GTP hydrolysis of SRP and SRP receptor, nascent polypeptide and ribosome transferred to cytoplasmic side of this protein complex
* has several ER integral membrane protein subunits forming an hourglass shaped aqueous channel
36
Sec61 translocon aqueous channel
* contains "pore ring" of six hydrophobic amino acids at the narrowest diameter of the channel
* gate to seal channel to ions and small molecules
* block by a short a-helix plug (2nd gatekeeping mechan
37
Signal peptidase
N-terminal signal sequence is cleaved by this as it enters the ER lumen during co-translational translocation
38
transmembrane domain
* stretch of 16-25 hydrophobic residues
* anchor protein in membrane bilayer
39
single-pass integral membrane proteins
type I, II, III and tail-anchored membrane proteins
40
type I membrane protein
* N(ER lumen) - C (cytosol) = final orientation
* polypeptide-ribosome complex targets to and associates with translocon
* Eventually **first and only** hydrophobic TMD enters transolocon
* TMD = stop-transfer anchor (STA) sequence
* STA moves out of translocon and translation continues until it completes
41
Type II membrane protein
* N(cytosol) - C(ER lumen) = final orientation
* no N-terminal signal sequence
* internal signal-anchor (SA) sequence
* **first and only** TMD functions as **signal sequence for SRP binding AND mediating polypeptide-ribosome complex targeting to translocon**
* SA enters translocon
* SA flipped in translocon so that N-terminus faces cytosol
* orientation mediated by positively charge AAs upstream of SA
* positive-outside rule
42
Type III membrane protein
* N(ER lumen) - C(cytosol)
* no N-terminal signal sequence
* internal signal-anchor (SA) sequence
* SRP-dependent translocon targeting
* Positively charged AAs downstram of SA
* SA not flipped
43
multi-spanning integral membrane proteins
* type IV ER membrane proteins
* multiple TMDs
* no N-terminal signal sequence
* BOTH internal SA sequences (serve to target protein to ER in SRP-dependent manner and
anchor protein in ER membrane depending on positive-outside rule)
* internal STA sequences
(serve to stop transfer of protein through and anchor into ER membrane)
44
stop transfer anchor (STA) sequence
stop transfer of protein through and anchor into ER membrane
45
Signal-Anchor Sequence (SA)
translocon recognizes positive residues of this sequence and flips (type II) or moves the TMD out of the translocon (type III)
46
Positive-outside rule
* positively charged residues adjacent to TMD
* determine orientation of TMD/membrane protein
47
membrane bilayer asymmetry
* **integral membrane proteins** – different regions of protein located on either cytoplasmic or
exoplasmic (i.e., ER luminal face of ER membrane)
* **peripheral membrane proteins** – located on either cytoplasmic or lumenal side of ER membrane
* **membrane phospholipids** - distributed unequally between cytoplasmic and exoplasmic leaflets of
ER membrane bilayer
48
glycosylation
covalent addition of carb side chains to specific amino acids of protein
49
glycoproteins
most proteins synthesized in ER are these type of proteins
50
N-linked glycosylation
* most common type of glycosylation
* addition of short sugar monomer chains to terminal amino group of asparagine
51
Core glycosylation
* various ER membrane-bound glycosyltransferases synthesize core oligosaccharide
* begins with addition of first sugar to dolichol phosphate
* glycosyltransferases continue to add sugars at specific positions on growing core oligosaccharide
52
Core modification
* second stage of N-linked glycosylation
* attached 14-sugar core oligosaccharide(s) sequentially ‘trimmed’ and ‘modified
* two (of 3) terminal glucose units removed (‘trimmed’) by ER lumenal glucosidases
* subsequent removal (and re-addition) of last glucose unit important for proper protein folding/assembly (i.e., quality control)
53
Glycosyltransferases
enzymes that synthesize core oligosaccharides and add sugars to growing core oligosaccharide
54
core oligosaccharides
highly branched oligosaccharide chain consisting of 14 sugar residues, including mannoses and 3-glucose-long terminal branch (important for protein quality control
55
dolichol phosphate
addition of first sugar to this molecule in core glycosylation
56
tunicamycin
blocks first step of N-linked glycosylation (inhibits glycosyl-
transferase action), preventing proper folding of nascent ER proteins
57
N-linked glycosylation motif
core oligosaccharide transferred to lumenal-facing portions of nascent ER proteins
with specific amino acid sequence motif: –N-x-S/T-
58
glucosidase
removes (‘trims’) last glucose unit from core oligosaccharide during
latter step in N-linked glycosylation process
59
ER protein quality control
ER-associated degradation (ERAD) and unfolded protein response (UPR) pathway
60
ER associated degradation pathway (ERAD)
* involves AAA ATPase p97
* ER membrane uses ATP hydrolysis to pull misfolded/misassembled proteins across ER membrane into cytosol
* in cytoplasm oligosaccharide chains removed and misfolded/misassembled protein poly-ubiquitinated
61
Unfolded Protein Response (UPR) pathway
* each pathway mediated by unique protein sensor:
* Ire1
* PERK
* ATF6
62
UGGT monitoring enzyme
* glucosyltransferase – serves as protein “conformation-sensing protein”
* recognizes hydrophobic residues usually ‘masked’ (buried) inside correctly-folded protein
* adds back single glucose unit to oligosaccharide core
63
ER protein degradation
* Proteasome - ‘barrel-shaped’, multi-subunit protein-degrading machine located in cytoplasm (and nucleus)
* oligosaccharide chains removed and misfolded/misassembled protein
poly-ubiquitinated
64
p97 AAA ATPase
ER membrane protein utilizes ATP hydrolysis to ‘pull’ misfolded/misassembled
proteins across ER membrane into cytosol
65
proteasome
barrel-shaped’, multi-subunit protein-degrading machine located in cytoplasm (and nucleus)
66
PERK-mediated URP pathway
* cytoplasmic-facing kinase domains of ‘activated’ dimer phosphorylate (inhibit) eIF2a (cytosolic protein translation factor required for initiation of protein synthesis – participates in ribosome-mRNA binding)
* decrease in cellular protein synthesis
67
ATF6-mediated UPR pathway
* ‘active’ - moves from ER to Golgi
* at Golgi, the cytoplasmic-facing, transcription factor domain is cleaved off by a Golgi-associated protease
* in nucleus, transcription factor domain upregulates genes encoding key proteins involved in ER quality control
68
ERES-derived transport vesicles
what properly-folded proteins are moved out of ER to Golgi and/or other compartments in endomembrane system in
69
COPI
* coat proteins
* move backwards from golgi to ER and backward within golgi
70
COPII
* coat proteins
* move forward from ERES to golgi
71
Clathrin
* coat proteins
* move from golgi to endosomes or from plasma membrane to endosomes
72
anterograde transport
forward transport
ERES -> golgi
73
retrograde transport
backward transport
golgi -> ERES
74
Sar1
* soluble COPII GTPase component
* Recruited from cytoplasm to ER via Sec12 binding
75
Sec12
* ER integral membrane protein
* GEF that catalyzes exchange of GDP for GTP on Sar1
76
Sec23 and Sec24
* recruited by Sar1
* act as structural scaffolding and promote initial outward bending of ERES membrane
77
ER export sorting signal
* mediates selection of vesicle membrane proteins by Sec24
78
Sec13 and Sec 31
* self-assemble into outer, cage-like lattice and act as structural
‘outer scaffolding’ for growing COPII vesicle bud
* promote additional outward bending
79
cis-golgi network
* vesicles traffic from ERES here
* incoming vesicles fuse with one another to form this
* interconnected network of vesicles and tubules
80
Rab proteins
* large family of lipid-membrane-anchored, GTP-binding proteins
associated with all transport vesicles
* key regulators of vesicle trafficking and fusion
81
Rab effector proteins
‘activated’ Rab (Rab-GTP) binds to these specific proteins on target membrane
82
molecular bridge
unique vesicle Rabs associate with Rab effectors on specific target membranes
83
v-SNARE
* found on transport vesicle membranes
* incorporated into vesicle membrane at site of budding on ‘donor’
membrane compartment
84
t-SNARE
found on target ‘acceptor’ membranes
85
SNARE motif
* cytoplasmic-facing, coiled-coil domain in both v- & t-SNAREs that
extend from vesicle/target membrane surface
* they interact in v/t SNARES to form a stable SNARE complex
86
SNARE complex
* After ’docking’ of vesicle, SNARE protein interaction
brings membranes close together for fusion
* leads to membrane fusion
* pulls vesicle and target membrane close together
87
NSF and SNAP
* cytosolic (soluble) proteins
* bind SNARE complexes and unwind (via ATP hydrolysis) SNARE domains linking v/t-SNAREs (SNARE complex disassembly
88
ER retrieval sorting signals
‘escaped’ ER resident proteins returned from
CGN back to ER (retrograde transport) with this
89
KDEL
* sequence that most resident soluble ER proteins possess on their C-terminal
90
KDEL receptor
* integral transmembrane protein - lumenal-facing domain
binds to -KDEL sequence of ‘escaped’ soluble ER proteins
in CGN lumen
* cytoplasmic-facing domain recognized by COPI protein coat
91
C-terminal dilysine sequence
specific sequence that most resident ER membrane proteins for ER retrieval sorting (-KKxx-)
92
Reticuloplasmins
* ER molecular chaperones (BiP, calreticulin, calnexin)
* bind reversibly to ER proteins to prevent misfolding or aggregation
