Lecture 10: Lysosome Biogenesis Flashcards
Lysosomes are _____
heterogenous group of membranous organelles containing > 50 types of acid hydrolases (enzymes) that degrade macromolecules
lysosomes are involved in recycling so ___
breakdown products can be transported to cytosol for reuse
lysosomal enzymes have pH optimum of _____ compared to the cytoplasm’s ___
- 5
- 7.2
pH of lysosome is maintained by ____
proton pump (membrane protein complex with ATPase activity)
pH of lysosomes keeps its enzymes from ___
being active in the cytosol and digesting the cell
size of lysosomes ____
varies in zie (10-fold)
the size of lysosomes reflects ____
variety of digestive functions of intracellular and extracellular debris
lysosomes contain ____ which form dense precipitate in the presence of lead
-acid phosphatase
lysosomal storage disorders are ____
group of genetic disease in which people have reduced levels of lysosomal enzymes
(more enzymes missing, the worse the disease)
lysosomal storage disorders life expectancy
<15 years
lysosomal storage disorders inheritance pattern
autosomal recessive
lysosomal storage disorders result
lysosome cannot perform its recycling function and becomes clogged with proteins, lipids, and carbohydrates than can not be degraded
lysosomal storage disorders (Tay Sachs)
-lack of hexosaminidase (hydrolyzes GM2)
-progressive neurodegeneration
lysosomes + heart health
-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
lysosomes + covid 19
-virus deacidify lysosomes to exit cell via lysosomal trafficking
3 major pathways for entry into lysosomes for degradation of intracellular + extracellular material
1) endocytosis
2) phagocytosis
3)autophagy
endocytosis (def.)
-receptor mediated uptake of proteins and lipids at cell surface
-endocytosed material is delivered to lysosomes via endosomal system
phagocytosis (def.)
engulfment of large particles(bacteria + debris) at cell surface + delivery to lysosomes directly
autophagy (def.)
ER-derived membrane wrap round organelles to form autophagosomes
phagocytosis + autophagy have this in common
-don’t used endosomes as transport intermediate
endosomes (def.)
membranous organelles with acidic lumens that receive and sort cargo from TGN + endocytic cargo from PM
early endosomes (ee) are found _____ and receive cargo from ____
-near PM
-PM primarily
late endosomes (LE)/ multi-vesicular bodies are found ___ and receive cargo from ___
-near nucleus/golgi
-early endosomes +TGN, contain internal vesicles
newly-synthesized lysosomal proteins have multiple ____
pathways for delivery into lysosomes
many but not all lysosomal enzymes (soluble) are modified by ____
addition of M6P
____ contain membrane proteins
lysosomes
Glycoproteins destined for lysosomes are ____ in ER and contain the same ______ as non-lysosomal glycoproteins
-N-glycosylated
-core oligosaccharides
generation of M6P-lysomal protein in Golgi
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
___ distinguishes between lysosomal enzymes & regulatory secretory proteins in Golgi by recognizing lysosomal hydrolases
N-acetylglucosamine (GlcNAc) phosphotransferase
in vitro findings: endo H treated lysosomal enzymes ______ to intact lysosomal enzymes
inhibit phosphorylation
in vitro findings: fragments of endo H treated lysosomal enzymes ______ to intact lysosomal enzymes
do not inhibit phosphorylation
in vitro findings: denatured lysosomal enzymes ______
are not phosphorylated properly
in vitro findings:
N-acetylglucosamine (GlcNAc) phosphotransferase
bind only to______
fully folded lysosomal enzymes
Lysosomal recognition determinant is _____
conformation/folding-dependent as opposed to linear sequence like KDEL motifs
cathepsin D is a ___
lysosomal hydrolyase
pepsinogen has no ___
natural glycosylation sites
only high mannose type glycans become ____
phosphorylated by Enzyme 1
Addition of M6P prevents further ____
processing of N-linked glycans to endo H resistant types
resistance to endo H means what about modification by M6P?
-evidence that N-linked glycans are not modified by M6P
secreted proteins are all ___
100% resistant to endo H
if its going to lysosomes, then it can’t be ___
secreted
M6P receptor column
-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)
M6P receptor column: CP2 -chimeric
shows us that the major lysosomal determinant is in C-terminus of CD
CD C-terminus has two ___
discontinuous regions that are required for the generation of M6P (region A and B)
cathepsin D (regions A & B) are rich in ___
lysine (this is absent in Pep)
lysines alone are not ___
lysosomal recognition component
what is total lysosomal recognition component of CD?
lysine 203 in region A and lysines (265-348) in region B(all of region B)
Enzyme 2 recognized folded region_____ + one lysine from region ____
-B
-A
If you disrupt folded structure of lysosomal protein, ___
it won’t bind to enzyme 1
GlcNAc phosphotransferase (Enzyme 1) recognizes a signal patch on lysosomal enzymes–involves lysine residue and _____
-Catalyzes transfer of GlcNAc phosphate (from UDP-N-acetylglucosamine donor) to mannose in 6’ position of N-linked oligosaccharide
GlcNAc phosphotransferase works where?
in cis-Golgi
- Phosphodiesterase (enzyme 2) removes _____
GlcNAc leaving mannose 6-P
Mannose 6-P is recognized by ____in ____
-mannose 6-P receptors (MPR)
-TGN
mannose 6-P receptors (MPR)
Transmembrane proteins that cycle between TGN,
endosomes and plasma membrane
two types of mannose 6-P receptors (MPR)
CD-MPR (cation-dependent)
CI-MPR (cation-independent) - LARGER
-both essential to transport to lysosomes
MPR directs incorporation into
clathrin coated vesicles (CCVs)
MPR binding site on ___
luminal sides of proteins
ligands of MPR dissociate in ____ due to ___
-endosomes due to low pH
MPRs contain multiple sorting signals in their cytoplasmic domains that _____
bind different adaptor/linker proteins
Both receptors (MPR) are required for delivery of all lysosomal enzymes: (2)
- Neither has exclusive affinity for specific lysosomal enzymes
- Receptors have similar and overlapping functions
type of targeting signal for lysosomal enzymes
Carbohydrate-dependent sorting i.e. M6P
Carbohydrate independent
Carbohydrate independent targeting signal discovered through I-Cell Disease (lysosomal storage disease)
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
Carbohydrate independent receptors (soluble lysosomal proteins)
Sortilin and LIMP-2 (they don’t bind to M6P)
Targeting of Lysosomal Membrane proteins is _____ process
carbohydrate-independent
Targeting of Lysosomal Membrane proteins: Sorting signals located in____ + type of signals
cytoplasmic domains
* Tyrosine-based motifs (YXXø)
* DiLeucine-based motifs»>LL + variations (IL)
Targeting of Lysosomal Membrane proteins with what adaptors?:
protein interacts with _____ (TGN/endosomes) or ____ (plasma membrane) followed by clathrin coat.
-GGAs/AP-1 (TGN/endosomes)
-AP-2 (plasma membrane)
Targeting of Lysosomal Membrane proteins: sorting can be direct from TGN (biosynethitc pathway): _______
or indirect via PM (endocytic pathway): _____
-TGN»>Late Endosomes»>Lysosomes
-TGN»>plasma membrane»>early endosomes»>late endosomes»>lysosomes
Late endosomes fuse with ______ or _____
-preexisting lysosomes or preexisting endolysosomes
Endolysosomes eventually mature into lysosomes as ______
hydrolases complete the digestion of their contents, which can include intralumenal vesicles.
