lect 8: cytoplasmic membrane systems

Question 1

what are the learning objectives of this lecture?

Answer 1

-compare the biosynthetic (secretory) and endocytic pathways
-compare the structures and functions of the RER and SER, and their roles in the maintenance of cellular proteins and membranes
-describe the synthesis of secretory, lysosomal proteins
-describe the synthesis of integral membrane proteins
-describe the role of the ER in membrane biosynthesis

Question 2

what is the endomembrane system?

Answer 2

membrane-dependent compartmentalization (collection of membrane enclosed organelles)

group of membranous organelles
-endoplasmic reticulum (ER)
-golgi apparatus
-endosomes
-lysosomes
-peroxisomes

general functions
-regulate protein trafficking
-metabolism

Question 3

what is the table of the membrane-enclosed organelles and their functions?

Answer 3

Question 4

what is the table of what each organelle takes up the volume in a whole cell?

Answer 4

Question 5

where is the endomembrane found and what does it do?

Answer 5

-dynamic, integrated network found only in eukaryotic cells (bc have membrane bound organelles)

shuttles materials from one part of the cell to another
-e.g. golgi complex to the plasma membrane
-transport vesicles: membrane bound and move in a directed manner

Question 6

what is some terminology that should be known about vesicle transport in the endomembrane system?

Answer 6

material transported from donor compartment to recipient

budding: bud from donor membrane compartment
-move via motor proteins: “tracks” formed by microtubules and microfilaments of cytoskeleton

fusion: fuse with membrane of acceptor compartment

Question 7

what are the two types of pathways in the endomembrane system?

Answer 7

biosynthetic pathway (secretory pathway):
-proteins synthesized in ER
-modified at golgi complex (for the specificity of their function)
-transported to various destinations

endocytic pathway:
-materials move from outer surface of the cell to compartments
-ex: endosomes, lysosomes

Question 8

what are the modes of secretion in the secretory pathway?

Answer 8

constitutive
-materials transported in secretory vesicles
-discharged continuously (at varying rates), e.g. formation of the ECM and plasma membrane

regulated
-materials stored/secreted in secretory granules
-discharged in response to stimulus (therefore controlled secretion)

examples of stimulus:
-endocrine cells: hormones
-pancreatic acinar cells: digestive enzymes
-neurons: neurotransmitters

Question 9

what is another example of polarized cell structure?

Answer 9

Question 10

what is the endoplasmic reticulum?

Answer 10

continuous with membrane of nuclear envelope

synthesis of:
-most lipids
-proteins to go to other organelles or plasma membrane

consists of:
-sac-like sheets (cisternae)
-branched tubules
-cisternal space
-RER is more flat that SER
-SER is more tubular, pipe-like network

Question 11

what is the graph of the ER in live mammalian cells?

Answer 11

Question 12

what are the two parts of the endoplasmic reticulum?

Answer 12

rough ER (RER)
-bound ribosomes
-helicoidal membranes connect cisternae
-continuous with nuclear envelope

smooth ER (SER)
-NO ribosomes
-highly curved membranes so therefore a tubular network
-continuous with RER

Question 13

what is the shape/form of the rough ER?

Answer 13

helicoidal ramps

Question 14

what are the functions of the smooth ER?

Answer 14

1. steroid hormone synthesis in endocrine cells
   -gonads (sex hormones)
   -adrenal cortex (cortisol)
2. detoxification in liver
   -oxygenases
   -cytochrome P450 family
3. calcium ion sequestration, regulated release
   -sarcoplasmic reticulum (only called this inside muscle cells because calcium triggers contraction)

Question 15

what is the main function of the rough ER?

Answer 15

-free ribosome=in cytosol; proteins involved in specific functions are transported
-bound=specific set of proteins, mainly secretory vesicles

protein synthesis happens in a specific fashion of co-translation translocation
-translation: synthesis of proteins
-translocation: move from one place to another
-synthesis in RER and moved in RER at the same time

-polyribosomes=attached to same RNA strand

Question 16

what is the synthesis of proteins on membrane-bound bs free ribosomes?

Answer 16

sequence of amino acids in N-terminal portion of polypeptide determines synthesis site
-signal sequence
-6-15 hydrophobic amino acid residues (quite short)
-directs nascent polypeptide and ribosome to ER membrane

signal hypothesis
-proteins have “built in” address codes

Question 17

how did they discover what the signal sequence do?

Answer 17

removed signal sequence and transferred

Question 18

what is the table of some typical signal sequences?

Answer 18

dont memorize
-vary in length and amino acids
-made of hydrophobic, positive and negatively charged amino acids
-substitution can occur (negative can replace negative and will still have same function)

Question 19

what is the graph of the free ribosome cycle and the membrane-bound ribosome cycle?

Answer 19

Question 20

what is the synthesis of secretory or lysosomal proteins (soluble proteins)?

Answer 20

signal recognition particle (SRP) recognizes signal sequence
-binds polypeptide and ribosome which arrests synthesis (until we get to the ER)

complex is recruited to ER membrane
-interaction between SRP and SRP receptor on ER membrane

-ribosome “handed off” to translocon (channel protein in ER membrane)
-signal sequence recognition so polypeptide in inserted into translocon
-polypeptide synthesis resumes
-co-translational translocation deposits protein into ER lumen

Question 21

what is the released of signal recognition sequence by the SRP?

Answer 21

several steps in secretory protein synthesis/trafficking regulated
-GTP binding (requires input of energy)
-GTP hydrolysis

GTP (guanosine triphosphate)
-hydrolysis means energy

SRP+ SRP receptor are G-proteins
-GTP bound=interact
-GTP hydrolysis=triggers release

Question 22

what happens in the last step of synthesis of secretory or lysosomal proteins (soluble proteins)?

Answer 22

-signal peptidase: removes signal sequence
-oligosaccharyltransferase: adds carbohydrates (glycosylation)
-integral membrane proteins associated with translocon

-most proteins synthesized in ER end up as glycoproteins (adds sugars via oligo)

Question 23

what is the synthesis of integral membrane proteins of single pass?

Answer 23

-hydrophobic transmembrane segment shunted from translocon into bilayer
-single-spanning membrane proteins do not change their orientation (i.e. cytosolic portion will always remain in cytosol)

Question 24

what is the synthesis of integral membrane proteins of multi-spanning protein?

Answer 24

multi-spanning protein (many transmembrane domains)
-sequential transmembrane segments have opposite orientation
-alternating stop-transfer and start-transfer sequences

-if water soluble, if fully passes the ER (is not transmembrane protein)

Question 25

what else occurs in the endoplasmic reticulum?

Answer 25

glycosylation -most RER-synthesized proteins become glycoproteins (glycosylated) glycosyltransferases -add sugars to oligosaccharides -each transfers a specific monosaccharide -sugar arrangement in chain depends on spatial localization of enzymes in assembly line

Question 26

what is N-glycosylation?

Answer 26

-core segment assembled on lipid carrier which is dolichol phosphate which is embedded in ER membrane sugars are added to dolichol phosphate one-by-one by glycosyltransferases (membrane-bound) -different glycosyltransferases add different sugars at each steps, 2 NAG, 9 mannose and then 3 glucose on oligosaccharide

Question 27

what do oligosaccharyl-transferase do in N-glycosylation?

Answer 27

-pre-assembled block transferred by ER enzyme oligosaccharyl-transferase (transfers whole unit of sugars) -transferred to certain asparagines of nascent polypetide (Asn becomes glycosylated by oligotrans)

Question 28

many proteins are glycosylated on _______ residues in the ER?

Answer 28

asparagine

Question 29

what is the glycosylation and quality control in the endoplasmic reticulum?

Answer 29

glycoprotein folding assessed by a conformation-sensing enzyme (UGGT protein or UDP=glucose: glycoprotein glycosyltransferase) misfolded proteins -tagged with glucose -mannose removal -transported to cytosol -destroyed to proteasomes (ER-associated degradation process)

Question 30

what is the graph of quality control?

Answer 30

misfolded proteins: UDP checks for correct function in ER of proteins -removal of glucose -calnexin=chaperone -then checked by UDP for expose hydrophobic residues -exported if folded correctly -problem seen by UGGT: adds glucose back, which signal calnexin that it isn't folded, so it will try to fold it again, then glucose is removed, checked by UDP again in cycles. If still not right, it will be targeted for destruction -mannose is removed, which triggers for transport to the cytosol so proteasomes will degrade to we can reuse amino acids

Question 31

what are the things involved in glycosylation and quality control?

Answer 31

glycosidase I and II remove 2 glucose -absence of terminal glucose signals that its ready to be checked for folding (look if hydrophobic amino acids are exposed) by UGGT -adds terminal glucose again (cycles)

Question 32

what happens to the proteins that are still misfolding in the glycosylation and quality control?

Answer 32

Question 33

what is the unfolded protein response in the ER?

Answer 33

-accumulation of misfolded proteins triggers unfolded protein response (UPR) protein sensors that monitor (unfolded/misfolded protein) -kept in inactive state by BiP (molecular chaperone) -increased accumulation causes BiP to be incapable of inhibiting sensors because they are recruited to help -activated sensors signal trigger proteins involved in destruction of misfolded proteins

Question 34

what is the graph of the unfolded protein response?

Answer 34

-sensors identify misfolded proteins -BiP binds to sensors when not many unfolded -when alot of unfolded, BiP relocated and is involved in trying to solve the problem -remove of BiP activates sensors, which triggers response to stop protein synthesis -phosphorylated proteins binds to ribosomes which halts synthesis

Question 35

what are the two pathways in the unfolded protein response?

Answer 35

activated sensors ->mult. signals -nucleus -cytosol pathway 1 (step 2) -PERK, elF2a (inhibits protein synthesis) pathway 2 (step 2a) -ATF6 which increases the amount of chaperones, coat proteins, and proteins of quality control machinery

Question 36

what are the two responses in the unfolded protein response?

Answer 36

stops protein synthesis (lets not make the situation worse) -PERK is a protein kinase and is inactivated by binding with BiP -PERK gets dimerized and then phosphorylates elF2a which triggers binding to small subunit of the ribosome which stops translation increases proteins to deal with problem -ATF6 is inactivated with the binding of BiP -activated means the translocation to the golgi complex, where it dissociates into cytosolic compartments, which is transported into the nucleus. This acts as transcription factor, which influences transcription of different genes. these genes have different functions so they increase the amount of chaperones, coat proteins, and proteins of quality control (like UGGT)

Question 37

what is the membrane biosynthesis in the endoplasmic reticulum?

Answer 37

-membranes arise from pre-existing membranes (adds, pinches then moves) -enzymatically modified as move from ER to other cellular compartments -asymmetry established in ER: cytosolic face, luminal/extracellular face

Question 38

how do we change lipid compositions of membrane biosynthesis of the endoplasmic reticulum?

Answer 38

-different organelles have different membrane lipid compositions change lipid composition by 1. enzymatic modification of lipid head group 2. preferentially including or excluding different phospholipids in forming vesicles 3. lipid transfer proteins