Bsci 420: Cell Biology Lectures 12-14

Front 1

Secretion process

Back 1

-Synthesis of proteins on ER and co-translational insertion

-Production of vesicles (formation of golgi cisterni)

-Golgi enzymes move countercurrent fashion

Front 2

Factors that allow proteins to interact with membrane receptors

Back 2

shape, size, charge

Front 3

Destination of post-golgi vesicles

Back 3

1. Plasma membrane: exocytosis and fusion (become integral membrane proteins)

2. Endosomes: formation of lysosomes

Front 4

v-snares and t-snares

Back 4

control fusion of membranes along with other docking proteins

Front 5

Discovery of lysosomes

Back 5

Isolated from livers, hypotonic lysing increases enzymatic activity of protein phosphatases --> special population of vesicles associated with lytic activity

Front 6

Protein phosphatase

Back 6

present in lysosomes and trans-golgi cisterni

Front 7

Primary and secondary lysosomes

Back 7

Termed by electron microscopists

Process later examined dynamically and determined to be continuous maturation

Shift terms to pre-lysosome and lysosome

Front 8

Determining destination of golgi-derived vesicle

Back 8

proteins within vesicle, receptors and proteins on vesicle membrane

Front 9

Hurler syndrome

Back 9

individual lacks capacity to make specific lysomal enzymes - lysosomes are swollen due to accumulated substrate

Front 10

Autophagy

Back 10

alternative to using lysosomes to degrade substrate

Wrapping substrate with ER and expelling

Front 11

Correcting hurler syndrome

Back 11

add missing enzyme to external space - it will reach its destination through endocytosis

can't really be used in vivo

Front 12

I cell deficiency

Back 12

single gene trait, lysosomal enzymes are all secreted --> swollen lysosomes

tx: addition of WT enzyme with mannose-6-phosphate tag to extracellular space

Front 13

Mannose-6-phosphate

Back 13

acts as competitive inhibitor of endocytosis

if terminal phosphate is removed - protein is secreted

raise MUT in presence: restore normal function

Front 14

Cis-golgi enzyme

Back 14

adds phosphorylated n-acetylglucosamine to mannose present on lysosomal proteins through esterification reaction

Front 15

Trans-golgi enzyme

Back 15

cleaves n-acetylglucosamine from phosphorylated mannose leaving mannose-6-phosphate tag on lysosomal protein

Front 16

M6P receptors

Back 16

bind vesicle proteins - move to endosome -dissociate during acidification and recycled to golgi

Front 17

M6P receptor recycling

Back 17

driven by retromers, site= trans golgi

Front 18

M6P tag

Back 18

marks vesicles as non-secreted

cleaved in functioning lysosome preventing return to golgi

Front 19

Post-translational import

Back 19

process of putting proteins into different compartments like mitochondria, chloroplast, peroxisomes and nucleus

Proteins synthesized in cytoplasm and incorporated as intact proteins

Front 20

Production of mitochondria/chloroplast proteins

Back 20

mainly occurs in cytoplasm (like 95-8%)

transfer proteins are responsible for targeting

Front 21

Co-translational import proteins

Back 21

not membrane associated, fold up after synthesis, no glycosylation

Front 22

HSP70

Back 22

example of carrier protein that bring cytoplasmic transcripts to target organelle

facilitates insertion of protein into mitochondrial matrix

Unfolds proteins on cytoplasmic side

Front 23

Simple version of post-translational import

Back 23

Protein binds to organelle, unfolds, threaded through membranes and refolded within appropriate compartment

Front 24

Target determination of post-translational import protein

Back 24

Determined by sequence of transcript and presence of specific membrane proteins that allow insertion to occur

Front 25

n-terminus

Back 25

responsible for insertion

penetrates membrane first through translocon

Front 26

comparison of outer membrane protein and inner membrane protein of mitochondria

Back 26

Differ in length of necessary sequence for targeting

Indicates that specific sequence does not encode target - sequence encodes SHAPE which determines target

Front 27

Common things to targeting motifs

Back 27

alpha-helical, positively charged, polarized hydrophobicity

Front 28

Cytochrome C

Back 28

ignores typical post-translational import pathway

CC1 typically inserted into mitochondria inner membrane

Front 29

Transit peptides in Cytochrome C

Back 29

Start transit peptide at n-terminus and immediately downstream is stop transit peptide

Front 30

Removal of CC1 stop transit peptide causes

Back 30

CC1 to be localized within the inner matrix

Front 31

Multiple sites of mitochondria protein insertion

Back 31

outer membrane, intermembrane space, inner membrane, matrix (default)

Front 32

Multiple sites of chloroplast protein insertion

Back 32

outer/inner membranes, intermembrane space, stroma membrane, thylakoid, stoma lamellae

Front 33

Start and stop transit peptides

Back 33

Determine site of insertion of post-translational import protein

Front 34

Post-translational import motifs are called...

Back 34

transit peptides

Front 35

Co-translational import motifs are called...

Back 35

transport peptides

Front 36

Endopeptidase

Back 36

removes transit peptides after insertion preventing movement out of the organelle

(cleavage causes change in conformation)

Front 37

TOM

TIM

Back 37

Translocon of outer and inner mitochondrial membranes respectively

align to allow protein to be inserted though both membranes at same time

Front 38

Why do TOM and TIM align?

Back 38

Prevent proteins from refolding before reaching matrix space

Front 39

TOC and TIC

Back 39

chloroplast counterparts of TOM and TIM, also align to allow proteins to pass into stromal space

Front 40

Inner membrane (of mitochondria)

Back 40

site of E generation using ATP synthase proteins and proton gradient

insertion of translocon pores does somewhat dissipate proton gradient

Front 41

Porins

Back 41

allow water to move through out membrane, inserted via TOM

prevented from travelling through inner membrane by chaperones in inner membrane space

Front 42

Mitochondria proteins that are not targeted to the matrix

Back 42

Either prevented from traveling through TIM (chaperones encourage protein folding) or are pulled into the matrix only to be pushed out again using stop transit peptide

Front 43

Treatment of mitochondria with ionophores

Back 43

Ionophores (DNP, CCCP) block import if protein hasn't yet started insertion

Block necessary energy requirements?

Front 44

Change conformation of proteins in matrix

Back 44

endopeptidase

sometimes allows for insertion into inner membrane

Front 45

ATP synthase activity

Back 45

required for continued insertion of proteins into mitochondria

Front 46

Dihydrofoliatereductrase

Back 46

cytoplasmic enzyme that creates color when exposed to a second enzyme

attach to start transit peptide: expressed in matrix

Front 47

Methotrexate

Back 47

binds to dihydrofoliatereductrase preventing functionality and unfolding - can't observe color in mitochonria

used to prove that protein unfolding is necessary for post-translation import

Front 48

New n-terminus

Back 48

generated after cleavage of start transit peptide

hydrophobic: insert into inner membrane or other points in cell/mitochondria

Front 49

Chloroplast post-translational import problems

Back 49

Not all membrane pairs have a site of energy production (like inner membrane of mitochondria)

Require different mechanisms for import

Front 50

Insertion into thylakoid

Back 50

secondary event mediated by SEC proteins, SRP and proton gradient driven pathway

Front 51

Peroxisomes

Back 51

Involved in oxidative rx (burn up toxic substances)

Major site of oxygen consumptions

Front 52

Disproven theories of peroxisomes

Back 52

must be generated from old peroxisomes

proteins arrive exclusively through post-translational import

Front 53

Peroxisome membrane proteins v. inner peroxisome proteins

Back 53

membrane: co-translation import but not through golgi

inner: post-translation import

Front 54

Products of peroxisome reactions

Back 54

Peroxide, phenols

ethanol to acid aldehyde

fatty acids to acetyl CoA

Front 55

Plant use of peroxisome

Back 55

breakdown of oils in seeds

Front 56

PEX proteins

Back 56

important in getting proteins into peroxisome

compose enzymes w/in peroxisome, channels proteins, chaperone proteins

Front 57

Peroxisome formation

Back 57

Fission: grow and divide (actin cables pull one into budding cell)

Front 58

Defective PEX

Back 58

Defects in PEX3, PEX 6 and PEX 19 --> no peroxisomes

Front 59

Cells without peroxisomes

Back 59

perform transvection with restorative proteins and peroxisomes are recovered

new peroxisomes can be generated from p-domain of smooth ER

Front 60

p-domain of Smooth ER

Back 60

PMP rich, bleb off as pre-peroxisomes and fuse together using snare proteins

Front 61

PMP proteins

Back 61

rich in p-domain of smooth ER

involved in peroxisome function and formation

Front 62

Dynamin and peroxisomes

Back 62

would form loop around budding section and cuts off new peroxisome from original (in fission)

DNM1 and VPS1 used instead

Front 63

DNM1 and VSP1

Back 63

used by peroxisomes instead of dynamin

DMN1 used for same function in mitochondria, similar alpha-helical structure to dynamin