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MCB2050 > Endomembrane System Part 1 > Flashcards

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

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2
Q

Donor Membrane Compartment

A

cargo-containing vesicle buds off this membrane compartment

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3
Q

Acceptor Membrane Compartment

A

vesicle transported to this membrane compartment

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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

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5
Q

Biosynthetic Pathway

A

materials transported from ER to golgi, endosomes and then lysosomes

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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

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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
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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
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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
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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
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11
Q

Subcellular Fractionation

A

isolation of organelles by centrifugation

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12
Q

homogenization

A
  • type of subcellular fractionation
  • cell/tissue disrupted while ensuring that organelles remain intact
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13
Q

homogenate

A
  • result of homogenization
  • gets filtered to remove unbroken cells and large fragments
  • subjected to differential centrifugation
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14
Q

supernatant

A

liquid at top of centrifuge tube

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15
Q

differential centrifugation

A

seperates intact organelles/cellular components of different size/density with increasing higher centrifugation speeds

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16
Q

Microsomes

A

fragments of ER membrane and/or plasma membrane that fuse and reform into small, spherical vesicles

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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
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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
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19
Q

liposomes

A
  • proteins are incubated with liposomes in cell-free systems
  • artificial, spherical vesicles consisting of phospholipid bilayer surrounding aqueous center
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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
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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
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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
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23
Q

Endoplasmic Reticulum lumen

A
  • aqueous space inside ER tubules and cisternae
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24
Q

Endoplasmic Reticulum Cisternae and Tubules

A
  • shapes mediated by reticulons
  • undergo bending, growth, shrinkage, fusion, fission
  • make the ER highly dynamic
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25
Q

Rough ER

A
  • subdomain of ER
  • mostly cisternae with bound ribosomes
  • protein and membrane phospholipid synthesis
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26
Q

Smooth ER

A
  • subdomain of ER
  • mostly curved tubules lacking ribosomes
  • Ca2+ storage and hormone synthesis
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27
Q

Mitochondria-Associated-Membranes and Plasma-Associated-Membranes

A
  • ER regions that make direct contact with mitochondria or plasma membrane
  • membrane protein and lipid exchange
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28
Q

ER Exit Sites

A

ER regions where transport vesicles bud off enroute to Golgi

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29
Q

Free Ribosomes

A
  • in cytoplasm
  • fate of nascent properly-folded soluble or membrane protein in cytoplasm: remain in cytoplasm OR target to proper intracellular component
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30
Q

ER Membrane-Bound Ribosomes

A
  • 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
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31
Q

Co-Translational Translocation

A
  • protein targeting to and across ER membrane
  • uses signal sequence, SRP, SRP receptor, Sec61 translocon, a-helix plug, signal peptidase
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32
Q

Signal Sequence

A
  • in Co-translational translocation:
  • a stretch of 8-15 hydrophobic amino acids on the N-terminus of the nascent growing polypeptide
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33
Q

Signal Recognition Particle

A
  • In co-translational translocation
  • recognizes exposed signal sequence on polypeptide
  • binds to ribosome and stops protein translation
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34
Q

SRP Receptor

A
  • in co-translational translocation
  • SRP complex binds to it
  • GTP hydrolysis releases the complex
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35
Q

Sec61 translocon

A
  • 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
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36
Q

Sec61 translocon aqueous channel

A
  • 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
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37
Q

Signal peptidase

A

N-terminal signal sequence is cleaved by this as it enters the ER lumen during co-translational translocation

38
Q

transmembrane domain

A
  • stretch of 16-25 hydrophobic residues
  • anchor protein in membrane bilayer
39
Q

single-pass integral membrane proteins

A

type I, II, III and tail-anchored membrane proteins

40
Q

type I membrane protein

A
  • 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
Q

Type II membrane protein

A
  • 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
Q

Type III membrane protein

A
  • 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
Q

multi-spanning integral membrane proteins

A
  • 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
Q

stop transfer anchor (STA) sequence

A

stop transfer of protein through and anchor into ER membrane

45
Q

Signal-Anchor Sequence (SA)

A

translocon recognizes positive residues of this sequence and flips (type II) or moves the TMD out of the translocon (type III)

46
Q

Positive-outside rule

A
  • positively charged residues adjacent to TMD
  • determine orientation of TMD/membrane protein
47
Q

membrane bilayer asymmetry

A
  • 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
Q

glycosylation

A

covalent addition of carb side chains to specific amino acids of protein

49
Q

glycoproteins

A

most proteins synthesized in ER are these type of proteins

50
Q

N-linked glycosylation

A
  • most common type of glycosylation
  • addition of short sugar monomer chains to terminal amino group of asparagine
51
Q

Core glycosylation

A
  • 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
Q

Core modification

A
  • 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
Q

Glycosyltransferases

A

enzymes that synthesize core oligosaccharides and add sugars to growing core oligosaccharide

54
Q

core oligosaccharides

A

highly branched oligosaccharide chain consisting of 14 sugar residues, including mannoses and 3-glucose-long terminal branch (important for protein quality control

55
Q

dolichol phosphate

A

addition of first sugar to this molecule in core glycosylation

56
Q

tunicamycin

A

blocks first step of N-linked glycosylation (inhibits glycosyl-
transferase action), preventing proper folding of nascent ER proteins

57
Q

N-linked glycosylation motif

A

core oligosaccharide transferred to lumenal-facing portions of nascent ER proteins
with specific amino acid sequence motif: –N-x-S/T-

58
Q

glucosidase

A

removes (‘trims’) last glucose unit from core oligosaccharide during
latter step in N-linked glycosylation process

59
Q

ER protein quality control

A

ER-associated degradation (ERAD) and unfolded protein response (UPR) pathway

60
Q

ER associated degradation pathway (ERAD)

A
  • 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
Q

Unfolded Protein Response (UPR) pathway

A
  • each pathway mediated by unique protein sensor:
  • Ire1
  • PERK
  • ATF6
62
Q

UGGT monitoring enzyme

A
  • 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
Q

ER protein degradation

A
  • Proteasome - ‘barrel-shaped’, multi-subunit protein-degrading machine located in cytoplasm (and nucleus)
  • oligosaccharide chains removed and misfolded/misassembled protein
    poly-ubiquitinated
64
Q

p97 AAA ATPase

A

ER membrane protein utilizes ATP hydrolysis to ‘pull’ misfolded/misassembled
proteins across ER membrane into cytosol

65
Q

proteasome

A

barrel-shaped’, multi-subunit protein-degrading machine located in cytoplasm (and nucleus)

66
Q

PERK-mediated URP pathway

A
  • 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
Q

ATF6-mediated UPR pathway

A
  • ‘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
Q

ERES-derived transport vesicles

A

what properly-folded proteins are moved out of ER to Golgi and/or other compartments in endomembrane system in

69
Q

COPI

A
  • coat proteins
  • move backwards from golgi to ER and backward within golgi
70
Q

COPII

A
  • coat proteins
  • move forward from ERES to golgi
71
Q

Clathrin

A
  • coat proteins
  • move from golgi to endosomes or from plasma membrane to endosomes
72
Q

anterograde transport

A

forward transport
ERES -> golgi

73
Q

retrograde transport

A

backward transport
golgi -> ERES

74
Q

Sar1

A
  • soluble COPII GTPase component
  • Recruited from cytoplasm to ER via Sec12 binding
75
Q

Sec12

A
  • ER integral membrane protein
  • GEF that catalyzes exchange of GDP for GTP on Sar1
76
Q

Sec23 and Sec24

A
  • recruited by Sar1
  • act as structural scaffolding and promote initial outward bending of ERES membrane
77
Q

ER export sorting signal

A
  • mediates selection of vesicle membrane proteins by Sec24
78
Q

Sec13 and Sec 31

A
  • self-assemble into outer, cage-like lattice and act as structural
    ‘outer scaffolding’ for growing COPII vesicle bud
  • promote additional outward bending
79
Q

cis-golgi network

A
  • vesicles traffic from ERES here
  • incoming vesicles fuse with one another to form this
  • interconnected network of vesicles and tubules
80
Q

Rab proteins

A
  • large family of lipid-membrane-anchored, GTP-binding proteins
    associated with all transport vesicles
  • key regulators of vesicle trafficking and fusion
81
Q

Rab effector proteins

A

‘activated’ Rab (Rab-GTP) binds to these specific proteins on target membrane

82
Q

molecular bridge

A

unique vesicle Rabs associate with Rab effectors on specific target membranes

83
Q

v-SNARE

A
  • found on transport vesicle membranes
  • incorporated into vesicle membrane at site of budding on ‘donor’
    membrane compartment
84
Q

t-SNARE

A

found on target ‘acceptor’ membranes

85
Q

SNARE motif

A
  • 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
Q

SNARE complex

A
  • 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
Q

NSF and SNAP

A
  • cytosolic (soluble) proteins
  • bind SNARE complexes and unwind (via ATP hydrolysis) SNARE domains linking v/t-SNAREs (SNARE complex disassembly
88
Q

ER retrieval sorting signals

A

‘escaped’ ER resident proteins returned from
CGN back to ER (retrograde transport) with this

89
Q

KDEL

A
  • sequence that most resident soluble ER proteins possess on their C-terminal
90
Q

KDEL receptor

A
  • 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
Q

C-terminal dilysine sequence

A

specific sequence that most resident ER membrane proteins for ER retrieval sorting (-KKxx-)

92
Q

Reticuloplasmins

A
  • ER molecular chaperones (BiP, calreticulin, calnexin)
  • bind reversibly to ER proteins to prevent misfolding or aggregation

MCB2050 (5 decks)

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