Lecture 6: Lysosomes - Structure, Regulation, and Function

Comprehensive vocabulary flashcards generated from lecture notes covering lysosomal components, biogenesis, trafficking, nutrient sensing signaling pathways, membrane repair, lysophagy, and analytical techniques.

Lysosome

The major degradative compartment of eukaryotic cells, characterized by an acidic lumen ($\text{pH} \times 4.5\text{--}6.0$), which orchestrates nutrient sensing, macromolecular breakdown, recycling, repair, and stress adaptation.

Three protein classes for lysosome function

1. Lumenal proteins (at acidic pH)

2. Integral membrane porteins

3. Membrane associated proteins

Lumenal proteins

• ~60 hydrolases mediate degradation.

• Saposins and granulins participate in macromolecular breakdown. ( degradation)

Integral membrane proteins

• Permeases and ion channels (transport nutrients).

• V-ATPase: Responsible for acidification.

Nutrient sensors: SLC38A9, LYCHOS

Structural proteins: LAMPs, LIMPs

Integral proteins- (SLC38A9, LYCHOS)

SLC38A9 - amino acid

LYCHOS - cholesterol

Both are nutrient sensors

Membrane associated proteins

Signaling proteins: mTORC1, Rag GTPases

• Transcription factors: MiT/TFE (dynamically interact with membrane).

Acidic Hydrolases

A group of approximately 60 lumenal enzymes (including proteases, lipases, nucleases, glycosidases, and phosphatases) that digest macromolecules and operate optimally under acidic conditions.

Saposins and Granulins

Lumenal cofactors within the lysosome that assist acidic hydrolases in the breakdown of macromolecules.

V-ATPase - acidification

An integral membrane proton pump that hydrolyzes ATP to pump $H^+$ ions into the lysosomal lumen, creating and maintaining its acidic environment.

ClC-7 - acidification

A lysosomal membrane channel that provides counter-ion transport to help maintain the pH gradient generated by V-ATPase.

LAMPs and LIMPs

Lysosome-Associated Membrane Proteins and Lysosomal Integral Membrane Proteins that serve as key structural integral membrane proteins of the lysosome.

Lysosome Composition and Functions

Mannose-6-phosphate (M6P) Receptor Pathway

The primary pathway for delivering soluble lysosomal hydrolases synthesized in the ER and sorted through the Golgi to lysosomes via clathrin-coated vesicles.

I-cell Disease

A condition caused by a defect in GNPT that prevents proper M6P tagging, resulting in the mis-targeting of lysosomal enzymes and their hyper-secretion outside the cell.

Lysosomal Storage Disorders (LSDs)

A class of diseases caused by defects in lysosomal function (e.g., GNPT mutations leading to mucolipidosis II and III) that result in the accumulation of undigested substrates.

mTORC1

Mechanistic target of rapamycin complex 1; a central signaling complex located on the lysosomal membrane that responds to nutrients, growth factors, energy status, and cellular stress to drive cell growth and proliferation.

Rag GTPases - mTORC1 recruit mechanism

Small GTPases functioning as heterodimers (Rag A/B and Rag C/D) that sense nutrient availability and recruit mTORC1 to the lysosomal surface under high nutrient conditions.

RHEB GTPase

A small GTPase that responds primarily to growth factor signaling and directly activates the kinase activity of mTORC1.

MiT/TFE Transcription Factors

A family of transcription factors (including MITF, TFEB, TFE3, and TFEC) that regulate lysosome biogenesis and autophagy by translocating to the nucleus in response to low nutrients or dephosphorylation.

how MiT / TFE factors work - 4 ways

1. MiT / TFE recognize CLEAR elements

2. Nuclear translocation - MiT / TFE can move to the nucleus and activate transcription of lysosomal and autophagy-related genes.

3. Regulation by mTORC1 ( high vs. low nutrients)

4. transcriptional activation - nuclear signaling promotes lysosomal biogenesis and function.

CLEAR Elements

Coordinated Lysosomal Expression and Regulation DNA sequence elements located in the promoter regions of many lysosomal genes, recognized and bound by MiT/TFE factors.

• when recognized to drive transcription under nutrient deficiency.

mTORC1 relationship with MiT/TFE under high and low nutrient conditions

• High conditions: mTORC1 inhibits MiT/TFE factors reducing lysosome biogenesis

• low nutrient conditions, MiT/TFE factors are released to promote lysosomal gene expression (mTORC1 inhibited)

under high nutrients what happens with mTORC1 and lysosomal biogenesis?

• mTORC1 is active and inhibits MiT/TFE factors, trapping them in the cytoplasm so they cant enter nucleus.

• therefore cant reach CLEAR (transcription factors)

• leading to reduced lysosomal biogenesis.

  • cells already had plenty of nutrients so therefore no more lysosomes needed

Do lysosomes move bidirectionally?

yes - through kinesin and dynein

Rab7 & Ar18b

regulate lysosome positioning and fusion with other organelles, playing a crucial role in lysosomal trafficking.

Kinesin (Lysosomal Transport)

A motor protein that mediates anterograde (peripheral) movement of lysosomes along microtubules.

Dynein (Lysosomal Transport)

A motor protein complex that mediates retrograde (perinuclear) movement and clustering of lysosomes along microtubules.

ESCRT Pathway (Lysosomal Repair)

• calcium-dependent,

• restores lysosomal integrity through inward constriction of damaged membrane edges.

PITT Pathway

• Phosphoinositide-dependent membrane tethering pathway

• coordinates lipid (cholesterol) transfer between the ER and lysosome to repair damaged lysosomal membranes.

When lysosomes are damaged what are the 2 repair pathways?

1. PITT pathway - takes longer b/c of transport involved

2. ESCRT pathway - more rapid

Lysophagy (damage > repair)

The selective autophagic degradation of damaged lysosomes occurs when membrane damage exceeds cellular repair capacity.

• Damaged lysosomes expose glycosylated proteins

Galectins (Lysophagy) - signal

Cytosolic proteins that bind to exposed glycosylated lysosomal lumenal proteins upon membrane rupture to signal ubiquitin ligase recruitment and initiate lysophagy.

• Galectin - signal

• Ubiquitin - ligase recruitment and lysophagy initiation.

• Autophagosome recruited - marks destruction

2 forms of lysophagy

1. lysophagy - canonical clearance: uses typical autophagy machinery (ligase, ubiquitin, galectins)

2. Non-canonical lysophagy: different autophagy pathway involving ATG proteins

Autophagic Lysosome Reformation (ALR)

• Restore lysosomes after lysophagy

• The process of restoring functional lysosome populations following autophagy through $PtdIns(4,5)P_2$ accumulation, kinesin-mediated tubulation, and proto-lysosome budding.

TBC1D15 (ALR Context)

A protein that acts alongside ATG8 proteins and LIMP2 to provide a scaffold for ALR machinery, promoting lysosomal tubule formation and regeneration.

LYTACs (manipulation strategy)

Lysosome targeting chimeras; a targeted degradation technology that utilizes lysosomal degradation pathways to selectively degrade target proteins.

Magnetic Isolation (Lysosomes) - Isolation & Analysis technique

An isolation strategy (such as NANOLYSE) using endocytosed iron dextran particles (FeDex) or superparamagnetic iron oxide nanoparticles (SPIONs) to pull out intact lysosomes using a magnet.

Lysosome interaction with ER, Mitochondria and Autophagosome

ER - allow lipid transfer and helps with calcium mobilization through STARD3. PITT pathways helps transfer lipids

mitochondria - help mark specific sites of mitochondrial fission and help with iron transfer

Autophagosome- lysosomes fuse with autophagosomes to degrade and recycle cytoplasmic components.

Types of Isolation & Analysis Techniques

1. Density Gradient Fractionation

2. Magnetic Isolation

3. Immunoprecipitation - antibody based isolation

Types of Manipulation Strategies

1. Pharmacological Modulation - use drugs to change lysosome

2. Genetic Approach - MiT/FEB manipulate genes

3. Targeted Degradation

Lysosomes and Health

1. Neuronal Health - lysosomes degrade damaged cells

2. Liver metabolism - during fasting the liver increases lysosomes biogenesis

3. Muscle health - excersisise promotes lysosomal activity and autophagy, enhancing muscle function.

4. Cancer cells - increase lysosomal degradation