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MCB*2050 Exam > Lecture 8 > Flashcards

Lecture 8 Flashcards

1
Q

Membrane biosynthesis at ER

A
  • membranes do not form de novo - all membranes arise from pre-existing membranes
  • most membrane proteins and lipids synthesized at ER
    exceptions: glycolipids synthesized in the Golgi and unique chloroplast and mitochondrial proteins & lipids
  • nascent ER membrane proteins & lipids can traffic to other cellular membranes
    e.g., move to other ER subdomains (via lateral diffusion through bilayer) OR other ‘downstream’ organelles of endomembrane system (via transport vesicles)
  • results in each organelle possessing unique complement of membrane proteins & lipids
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2
Q

integral membrane proteins –

A

different regions of protein located on either cytoplasmic or exoplasmic (i.e., ER luminal face of ER membrane)

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3
Q

peripheral membrane proteins

A

located on either cytoplasmic or lumenal side of ER membrane

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4
Q

membrane phospholipids -

A

distributed unequally between cytoplasmic and exoplasmic leaflets of ER membrane bilayer

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5
Q

final steps in co-translational translocation pathway involve ‘processing’ of newly- synthesized (nascent) protein in ER lumen

A
  1. signal sequence cleavage - removal of N-terminal signal sequence by signal peptidase
  2. initial stages of glycosylation – covalent addition of unique carbohydrate side chains to specific amino acids of nascent protein (required for proper folding, protein-protein binding, etc)
  3. protein folding and assembly – nascent protein folded into proper 3D conformation and oligomeric assembly by molecular chaperones (reticuloplasmins)
  4. protein quality control – misfolded and/or improperly assembled proteins recognized and degraded
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6
Q

ER serves as an ideal …

A

processing and quality control site for nascent proteins since represents first compartment in endomembrane system (i.e., biosynthe

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7
Q

What type of proteins are
synthesized in ER?

A

most proteins (soluble and membrane) synthesized in ER are glycoproteins

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8
Q

Most common type of glycosylation

A

N-linked glycosylation :
addition of specific short chains of sugar monomers (linked together in specific order to form
oligosaccharide) to terminal amino group of asparagine (N)

*two stages:
i) core glycosylation
ii) core modification

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9
Q

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

A

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

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10
Q

Core glycosylation (stage 1)

A

*various ER membrane-bound glycosyltransferases synthesize core oligosaccharide
* begins with addition of first sugar to dolichol phosphate
membrane lipid serving as membrane ‘anchor’ and ‘carrier’ for new, growing core oligosaccharide
* glycosyltransferases continue to add sugars at specific positions on growing core oligosaccharide

  • final step… transfer of core oligosaccharide from dolichol lipid carrier to nascent soluble/membrane protein while being synthesized (via Sec61 co-translational translocation pathway)
    ‘empty’ dolichol phosphate recycled for another round of core oligosaccharide synthesis
  • core oligosaccharide transferred to lumenal-facing portions of nascent ER proteins
    with specific amino acid sequence motif: –N-x-S/T-
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11
Q

Core modification (stage 2)

A
  • attached 14-sugar core oligosaccharide(s) sequentially ‘trimmed’ and ‘modified’
  • two (of 3) terminal glucose units removed (‘trimmed’) by ER lumenal glucosidases (Step 1 & 2)
  • subsequent removal (and re-addition) of last glucose unit (Step 3 & 3a) important for proper protein folding/assembly (i.e., quality control)
  • during N-linked glycosylation and modification, nascent protein rapidly folded into proper 3D conformation
  • mediated by several ER lumen and membrane proteins - reticuloplasmins and protein disulfide isomerase (PDI)
    *core oligosaccharide(s) added to nascent protein during N-linked glycosylation also contribute to proper protein folding/assembly and stability
    ….and participate protein quality control…
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12
Q

reticuloplasmins –

A

ER molecular chaperones, including BiP, calreticulin and calnexin bind transiently (reversibly) to nascent ER proteins to prevent misfolding or aggregation

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13
Q

protein disulfide isomerase (PDI) –

A

catalyzes formation of intra/intermolecular disulfide bonds disulfide bonds between cysteine residues on same or different nascent polypeptides promote proper folding and assembly by stabilizing their proper 3D conformation

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14
Q

ER protein quality control

A
  • reticuloplasmins (and PDI) bind to nascent glycoprotein (with one remaining glucose unit) while being synthesized via Sec61 co-translational translocation pathway
    help mediate proper protein folding, oligomeric assembly, stability, etc
  • ER lumen glucosidase removes (‘trims’) last glucose unit from core oligosaccharide during latter step in N-linked glycosylation process (Step 3)
  • nascent protein released from reticuloplasmins….
  • if protein (soluble or membrane-bound) is properly folded/assembled…
    One mannose unit removed (‘trimmed’) by ER lumen mannosidase (Step 4) then…functions as ER (subdomain) resident protein or
    *transported (via vesicles) from ER to Golgi (where N-linked glycosylation continues); then resides in Golgi or moves to other compartment(s) in endomembrane system
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15
Q

What happens when a protein is released from the reticuloplasmins misfolded/misassembled

A
  • recognized by UGGT ‘monitoring’ enzyme - glucosyltransferase – serves as protein “conformation-sensing protein”
    recognizes hydrophobic residues usually ‘masked’ (buried) inside correctly-folded protein
  • UGGT adds back single glucose unit to oligosaccharide core (Step 3a)
  • misfolded/misassembled protein binds (again) to calnexin, calreticulin and BiP
    mediate (again) proper protein folding, oligomeric assembly, etc
  • process continues (repeated) until protein properly folded/assembled
  • misfolded/misassembled (abnormal) proteins eventually degraded (~5-60 min)
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16
Q

What happens when a protein is released from the reticuloplasmins misfolded/misassembled

A
  • process continues (repeated) until protein properly folded/assembled
  • misfolded/misassembled (abnormal) proteins eventually degraded (~5-60 min)
17
Q

What is the AAA ATPase p97 involved in the ER-Associated Degradation (ERAD) pathway

A

AAA ATPase p97 – ER membrane protein utilizes ATP hydrolysis to ‘pull’ misfolded/misassembled proteins across ER membrane into cytosol

Retrotranslocation

18
Q

ER protein degradation

What happens in the cytoplasm?

A
  • in cytoplasm… oligosaccharide chains removed and misfolded/misassembled protein poly-ubiquitinated - protein linked to chain of repeating (poly) ubiquitin units

ubiquitin (UB) - small (76 amino acids) protein involved in diverse cellular functions

mono-UB – serves as ‘signal’ for targeting membrane proteins into intralumenal vesicles of
late endosomes/multivesicular bodies [see later]

poly-UB – serves as ‘signal’ for ER protein degradation and for most other cellular proteins
destined for normal turnover
* poly-UB protein degraded by proteasome

19
Q

What is a protosome and what are the steps in protosome degradation?

A

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

several steps in proteasome degradation process:
1. UB-protein binds to ‘cap’ or ‘lid’ of proteasome
2.Poly-UB chain removed (recycled)
3. protein ‘threaded’ into proteasome and degraded (via proteolysis)
4.‘free’ amino acids are reused for new protein synthesis

20
Q

Unders certain conditions, miss, folded/disassembled proteins can accumulate …

A

in ER to high levels

*overload ERAD protein degradation pathway
e.g., several diseases (CF, Alzheimer’s, etc) - misfolded proteins accumulate in ER and form toxic aggregates; leads to cell death

  • results in ‘ER stress’ - signals Unfolded Protein Response (UPR) pathways
  • each pathway mediated by unique protein sensor – ER integral membrane-spanning proteins e.g.,
  • Ire1 (Inositol-Requiring kinase 1) (Lodish Fig 13-2)
  • PERK (Protein kinase RNA-like ER Kinase)
  • ATF6 - (Activating Transcription Factor 6)
21
Q

ER protein quality control :
PERK and ATF6 UPR pathways

A
  • both membrane-bound PERK and ATF6 possess ER lumenal-facing ‘stress-sensing domains’
    bind to molecular chaperones (e.g, BiP) in ER lumen
  • in non-stress conditions, PERK and ATF6 sensors inactive by binding to BiP
  • in ER-stress conditions (increase in misfolded/misassembled proteins) UPR pathways activated…
22
Q

PERK-mediated UPR pathway

A
  • BiP released from PERK to aid in folding of accumulating (misfolded/misassembled) ER proteins
  • PERK dimerizes and becomes ‘active’
  • cytoplasmic-facing kinase domains of ‘activated’
    PERK dimer phosphorylate (inhibit) eIF2a cytosolic protein translation factor required for
    initiation of protein synthesis – participates in ribosome-mRNA binding
  • decrease in cellular protein synthesis (including at RER)
    molecular chaperones (e.g, BiP) available to focus on pre-existing (misfolded/misassembled) proteins in ER
  • ER stress is alleviated or (if not) cell death occurs
23
Q

ATF6-mediated UPR pathway

A
  • in ER-stress conditions, BiP released from ATF6
    BiP needed for folding accumulating (misfolded/misassembled) ER proteins
  • ‘active’ ATF6 moves from ER to Golgi (via transport vesicles from ER exit sites [see later])
  • at Golgi, the cytoplasmic-facing, transcription factor domain of ATF6 is cleaved off by a Golgi- associated protease
    *ATF6 transcription factor domain targets to nucleus via an exposed NLS (not exposed in full-length ATF6)
  • in nucleus, ATF6 transcription factor domain upregulates genes encoding key proteins involved in ER quality control:
    reticuloplasmins – assist in protein folding in ER (e.g., BiP)
    ER export components
    assist in moving (via ERES transport vesicles) properly- folded proteins out of ER to Golgi and/or other compartments in endomembrane system
    ERAD components – assist in degrading any remaining misfolded/misassembled proteins in ER
  • ER stress is alleviated or (if not) cell death occurs

MCB*2050 Exam (11 decks)

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