Lecture 10: Lysosome Biogenesis

Question 1

Lysosomes are _____

Answer 1

heterogenous group of membranous organelles containing > 50 types of acid hydrolases (enzymes) that degrade macromolecules

Question 2

lysosomes are involved in recycling so ___

Answer 2

breakdown products can be transported to cytosol for reuse

Question 3

lysosomal enzymes have pH optimum of _____ compared to the cytoplasm’s ___

Answer 3

• 5
• 7.2

Question 4

pH of lysosome is maintained by ____

Answer 4

proton pump (membrane protein complex with ATPase activity)

Question 5

pH of lysosomes keeps its enzymes from ___

Answer 5

being active in the cytosol and digesting the cell

Question 6

size of lysosomes ____

Answer 6

varies in zie (10-fold)

Question 7

the size of lysosomes reflects ____

Answer 7

variety of digestive functions of intracellular and extracellular debris

Question 8

lysosomes contain ____ which form dense precipitate in the presence of lead

Answer 8

-acid phosphatase

Question 9

lysosomal storage disorders are ____

Answer 9

group of genetic disease in which people have reduced levels of lysosomal enzymes
(more enzymes missing, the worse the disease)

Question 10

lysosomal storage disorders life expectancy

Answer 10

<15 years

Question 11

lysosomal storage disorders inheritance pattern

Answer 11

autosomal recessive

Question 12

lysosomal storage disorders result

Answer 12

lysosome cannot perform its recycling function and becomes clogged with proteins, lipids, and carbohydrates than can not be degraded

Question 13

lysosomal storage disorders (Tay Sachs)

Answer 13

-lack of hexosaminidase (hydrolyzes GM2)
-progressive neurodegeneration

Question 14

lysosomes + heart health

Answer 14

-you can get buildup of LDL that block major arteries (heart attack/coronary heart disease)
-cell have LDL receptors that bring cholesterol into cell to be degraded by lysosome

Question 15

lysosomes + covid 19

Answer 15

-virus deacidify lysosomes to exit cell via lysosomal trafficking

Question 16

3 major pathways for entry into lysosomes for degradation of intracellular + extracellular material

Answer 16

1) endocytosis
2) phagocytosis
3)autophagy

Question 17

endocytosis (def.)

Answer 17

-receptor mediated uptake of proteins and lipids at cell surface
-endocytosed material is delivered to lysosomes via endosomal system

Question 18

phagocytosis (def.)

Answer 18

engulfment of large particles(bacteria + debris) at cell surface + delivery to lysosomes directly

Question 19

autophagy (def.)

Answer 19

ER-derived membrane wrap round organelles to form autophagosomes

Question 20

phagocytosis + autophagy have this in common

Answer 20

-don’t used endosomes as transport intermediate

Question 21

endosomes (def.)

Answer 21

membranous organelles with acidic lumens that receive and sort cargo from TGN + endocytic cargo from PM

Question 22

early endosomes (ee) are found _____ and receive cargo from ____

Answer 22

-near PM
-PM primarily

Question 23

late endosomes (LE)/ multi-vesicular bodies are found ___ and receive cargo from ___

Answer 23

-near nucleus/golgi
-early endosomes +TGN, contain internal vesicles

Question 24

newly-synthesized lysosomal proteins have multiple ____

Answer 24

pathways for delivery into lysosomes

Question 25

many but not all lysosomal enzymes (soluble) are modified by ____

Answer 25

addition of M6P

Question 26

____ contain membrane proteins

Answer 26

lysosomes

Question 27

Glycoproteins destined for lysosomes are ____ in ER and contain the same ______ as non-lysosomal glycoproteins

Answer 27

-N-glycosylated -core oligosaccharides

Question 28

generation of M6P-lysomal protein in Golgi

Answer 28

1)N-acetylglucosamine (GlcNAc) phosphotransferase (Enzyme 1)- transfers GlcNac reside to mannose residue on lysosomal protein (phosphate bond) (with Uridine dpn GlcNAc ---> UMP) 2)phosphodiesterase (enzyme 2) removes GlcNac leaves M6P on lysosomal protein

Question 29

___ distinguishes between lysosomal enzymes & regulatory secretory proteins in Golgi by recognizing lysosomal hydrolases

Answer 29

N-acetylglucosamine (GlcNAc) phosphotransferase

Question 30

in vitro findings: endo H treated lysosomal enzymes ______ to intact lysosomal enzymes

Answer 30

inhibit phosphorylation

Question 31

in vitro findings: fragments of endo H treated lysosomal enzymes ______ to intact lysosomal enzymes

Answer 31

do not inhibit phosphorylation

Question 32

in vitro findings: denatured lysosomal enzymes ______

Answer 32

are not phosphorylated properly

Question 33

in vitro findings: N-acetylglucosamine (GlcNAc) phosphotransferase bind only to______

Answer 33

fully folded lysosomal enzymes

Question 34

Lysosomal recognition determinant is _____

Answer 34

conformation/folding-dependent as opposed to linear sequence like KDEL motifs

Question 35

cathepsin D is a ___

Answer 35

lysosomal hydrolyase

Question 36

pepsinogen has no ___

Answer 36

natural glycosylation sites

Question 37

only high mannose type glycans become ____

Answer 37

phosphorylated by Enzyme 1

Question 38

Addition of M6P prevents further ____

Answer 38

processing of N-linked glycans to endo H resistant types

Question 39

resistance to endo H means what about modification by M6P?

Answer 39

-evidence that N-linked glycans are not modified by M6P

Question 40

secreted proteins are all ___

Answer 40

100% resistant to endo H

Question 41

if its going to lysosomes, then it can't be ___

Answer 41

secreted

Question 42

M6P receptor column

Answer 42

-CD would bind (Run thru portion would contain Pep) -rinse with M6P (Bound fraction would contain CD0 -chimeric proteins with M6P would also bind (CP2)

Question 43

M6P receptor column: CP2 -chimeric

Answer 43

shows us that the major lysosomal determinant is in C-terminus of CD

Question 44

CD C-terminus has two ___

Answer 44

discontinuous regions that are required for the generation of M6P (region A and B)

Question 45

cathepsin D (regions A & B) are rich in ___

Answer 45

lysine (this is absent in Pep)

Question 46

lysines alone are not ___

Answer 46

lysosomal recognition component

Question 47

what is total lysosomal recognition component of CD?

Answer 47

lysine 203 in region A and lysines (265-348) in region B(all of region B)

Question 48

Enzyme 2 recognized folded region_____ + one lysine from region ____

Answer 48

-B -A

Question 49

If you disrupt folded structure of lysosomal protein, ___

Answer 49

it won't bind to enzyme 1

Question 50

GlcNAc phosphotransferase (Enzyme 1) recognizes a signal patch on lysosomal enzymes--involves lysine residue and _____

Answer 50

-Catalyzes transfer of GlcNAc phosphate (from UDP-N-acetylglucosamine donor) to mannose in 6’ position of N-linked oligosaccharide

Question 51

GlcNAc phosphotransferase works where?

Answer 51

in cis-Golgi

Question 52

2. Phosphodiesterase (enzyme 2) removes _____

Answer 52

GlcNAc leaving mannose 6-P

Question 53

Mannose 6-P is recognized by ____in ____

Answer 53

-mannose 6-P receptors (MPR) -TGN

Question 54

mannose 6-P receptors (MPR)

Answer 54

Transmembrane proteins that cycle between TGN, endosomes and plasma membrane

Question 55

two types of mannose 6-P receptors (MPR)

Answer 55

CD-MPR (cation-dependent) CI-MPR (cation-independent) - LARGER -both essential to transport to lysosomes

Question 56

MPR directs incorporation into

Answer 56

clathrin coated vesicles (CCVs)

Question 57

MPR binding site on ___

Answer 57

luminal sides of proteins

Question 58

ligands of MPR dissociate in ____ due to ___

Answer 58

-endosomes due to low pH

Question 59

MPRs contain multiple sorting signals in their cytoplasmic domains that _____

Answer 59

bind different adaptor/linker proteins

Question 60

Both receptors (MPR) are required for delivery of all lysosomal enzymes: (2)

Answer 60

* Neither has exclusive affinity for specific lysosomal enzymes * Receptors have similar and overlapping functions

Question 61

type of targeting signal for lysosomal enzymes

Answer 61

Carbohydrate-dependent sorting i.e. M6P Carbohydrate independent

Question 62

Carbohydrate independent targeting signal discovered through I-Cell Disease (lysosomal storage disease)

Answer 62

Cells lack GlcNAc phosphotransferase (enzyme 1) * Complete lack of lysosomal enzymes in most cell types – Lysosomal enzymes are secreted instead -However, MPRs function normally and can take up lysosomal enzymes with M6P via endocytic pathway -Cell type-specific: hepatocytes and lymphocytes have regular amount of enzymes

Question 63

Carbohydrate independent receptors (soluble lysosomal proteins)

Answer 63

Sortilin and LIMP-2 (they don't bind to M6P)

Question 64

Targeting of Lysosomal Membrane proteins is _____ process

Answer 64

carbohydrate-independent

Question 65

Targeting of Lysosomal Membrane proteins: Sorting signals located in____ + type of signals

Answer 65

cytoplasmic domains * Tyrosine-based motifs (YXXø) * DiLeucine-based motifs>>>LL + variations (IL)

Question 66

Targeting of Lysosomal Membrane proteins with what adaptors?: protein interacts with _____ (TGN/endosomes) or ____ (plasma membrane) followed by clathrin coat.

Answer 66

-GGAs/AP-1 (TGN/endosomes) -AP-2 (plasma membrane)

Question 67

Targeting of Lysosomal Membrane proteins: sorting can be direct from TGN (biosynethitc pathway): _______ or indirect via PM (endocytic pathway): _____

Answer 67

-TGN>>>Late Endosomes>>>Lysosomes -TGN>>>plasma membrane>>>early endosomes>>>late endosomes>>>lysosomes

Question 68

Late endosomes fuse with ______ or _____

Answer 68

-preexisting lysosomes or preexisting endolysosomes

Question 69

Endolysosomes eventually mature into lysosomes as ______

Answer 69

hydrolases complete the digestion of their contents, which can include intralumenal vesicles.