Cholesterol-dependent lysosomal docking of mTORC1 mediated by TM6SF1

This study identifies TM6SF1 as a cholesterol-bound lysosomal membrane protein that structurally stabilizes the Ragulator complex to facilitate mTORC1 docking, thereby linking lysosomal cholesterol levels directly to growth control and TFEB regulation.

The Gist

The Big Picture: The Cell's "Growth Switch" and Its Missing Key

Imagine your body's cells are like busy, high-tech factories. Inside these factories, there is a master control room called the lysosome. Its main job is to act as the recycling center, breaking down old parts and trash to make new materials.

But the lysosome does more than just clean; it also acts as a traffic cop for the factory's growth. It controls a giant switch called mTORC1.

• When mTORC1 is ON: The factory says, "We have plenty of food and energy! Let's grow and build new parts!"
• When mTORC1 is OFF: The factory says, "We are low on resources. Let's stop growing and start recycling everything to survive."

For years, scientists knew that the lysosome needed cholesterol (a type of fat) to turn this switch ON. But they didn't know how the cholesterol talked to the switch. This paper solves that mystery by introducing a new character: TM6SF1.

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The New Character: TM6SF1 (The Docking Station)

Think of the lysosome membrane as a busy bus terminal.

• mTORC1 is the bus that needs to arrive at the terminal to pick up passengers (nutrients) and start the engine (growth).
• The Ragulator Complex is the bus stop sign that tells the bus where to park.
• TM6SF1 is the docking station or the gatekeeper that holds the bus stop sign firmly in place so the bus can actually park.

The researchers discovered that TM6SF1 is a protein sitting on the lysosome wall. Its job is to grab the "bus stop sign" (Ragulator) and hold it tight. Without TM6SF1, the bus stop sign falls over, the bus (mTORC1) can't park, and the factory never turns on its growth mode.

The Secret Ingredient: Cholesterol as a "Structural Glue"

Here is the coolest part of the discovery. The researchers used a powerful microscope (Cryo-EM) to take a 3D picture of TM6SF1. They found that TM6SF1 isn't just a static gatekeeper; it has a special pocket inside it that holds a cholesterol molecule.

The Analogy:
Imagine TM6SF1 is a folding chair.

• When the chair is empty (no cholesterol), the legs are wobbly and the seat is floppy. It can't hold anything heavy.
• When you slide a cholesterol molecule into the seat (like a support brace), the chair suddenly becomes rigid, stable, and strong.

In the cell, cholesterol acts as this structural brace. It locks TM6SF1 into the perfect shape so it can grab the Ragulator complex and hold it steady.

• No Cholesterol? The chair collapses. TM6SF1 can't hold the bus stop sign. The growth switch stays OFF.
• With Cholesterol? The chair is solid. The bus parks. The growth switch turns ON.

What Happens When TM6SF1 is Missing?

The scientists created cells without TM6SF1 (like removing the gatekeeper from the bus terminal). Here is what happened:

1. The Bus Never Arrives: The mTORC1 "bus" couldn't dock at the lysosome.
2. The Factory Panics: Because the growth switch never turned on, the cell thought it was starving, even if it had plenty of food.
3. The Cleanup Crew Goes Wild: A protein called TFEB (think of it as the "Recycling Manager") usually stays in the breakroom (cytoplasm) when the factory is busy. But when the growth switch is broken, TFEB runs into the CEO's office (the nucleus) and screams, "We need to clean up everything!" This causes the cell to over-activate its recycling mode, which is not ideal for normal growth.

Why This Matters

This paper connects two major biological concepts that were previously thought to be separate:

1. Cholesterol Metabolism: How our bodies handle fats.
2. Cell Growth Control: How our bodies decide when to grow.

It turns out that cholesterol isn't just a fuel or a building block; it is a molecular key that physically locks the growth machinery into place.

In Summary:
The cell uses a protein called TM6SF1 as a docking station for its growth switch. To make this station work, it needs to be "glued" together by cholesterol. If you take away the cholesterol or break the protein, the growth switch gets stuck in the "off" position, and the cell gets confused, thinking it needs to clean up instead of grow. This discovery helps us understand how cells sense their environment and could lead to new treatments for diseases related to metabolism or growth.

Technical Summary

1. Problem Statement

Lysosomes serve as critical signaling hubs that regulate cell growth and metabolism via the mechanistic target of rapamycin complex 1 (mTORC1). While the recruitment of mTORC1 to the lysosomal surface is known to depend on the Ragulator complex (anchored by LAMTOR1) and amino acid sensing, the specific membrane proteins that facilitate the stable docking of the Ragulator-mTORC1 machinery and how lysosomal cholesterol integrates into this process remain unclear. Specifically, the physiological function of TM6SF1, a ubiquitously expressed lysosomal membrane protein with 10 transmembrane helices, was previously unknown. Unlike its homolog TM6SF2 (involved in hepatic lipoprotein lipidation), TM6SF1's role in cellular signaling had not been elucidated.

2. Methodology

The authors employed a multidisciplinary approach combining cell biology, biochemistry, structural biology, and computational modeling:

• Cellular Models: Generation of TM6SF1 knockout (KO) HEK293A cell lines using CRISPR/Cas9.
• Functional Assays:
  • Immunoblotting: Analysis of mTORC1 signaling markers (p70 S6K, 4E-BP1 phosphorylation) and autophagy markers (p62/SQSTM1).
  • Immunofluorescence: Visualization of TFEB (Transcription Factor EB) localization (nuclear vs. cytosolic) and colocalization studies with lysosomal markers (LAMP1/2) and Ragulator components.
  • Lysosomal Isolation: Use of LysoTag (TMEM192-2xFlag) to purify intact lysosomes for assessing mTORC1 recruitment.
  • Metabolic Probes: Assessment of lysosomal pH (pHLys Red/LysoPrime Green) and free cholesterol levels (Filipin staining).
• Biochemical Interaction Studies:
  • Co-Immunoprecipitation (Co-IP) & Pull-downs: To identify binding partners of TM6SF1 (specifically the Ragulator complex).
  • Cross-linking Mass Spectrometry: To map the interaction interface between TM6SF1 and LAMTOR1.
  • Lipid Analysis: Gas Chromatography–Mass Spectrometry (GC-MS) to identify lipids bound to purified TM6SF1.
• Structural Biology:
  • Cryo-Electron Microscopy (Cryo-EM): Determination of the human TM6SF1 structure at 2.9 Å resolution. To facilitate particle alignment, a construct (TM6SF1MBP) was engineered with an N-terminal Maltose-Binding Protein (MBP) and a C-terminal DARPin off7 fiducial marker.
• Computational Modeling: Molecular Dynamics (MD) simulations to assess the conformational flexibility of TM6SF1 in the presence and absence of cholesterol.

3. Key Contributions

• Identification of TM6SF1 Function: TM6SF1 is identified as a cholesterol-bound lysosomal docking factor essential for mTORC1 activation.
• Structural Elucidation: The first high-resolution (2.9 Å) structure of human TM6SF1, revealing it as a polytopic homodimer with a specific cholesterol-binding pocket.
• Mechanistic Link: Discovery of a direct physical interaction between TM6SF1 and LAMTOR1 (a subunit of the Ragulator complex), which stabilizes the Ragulator on the lysosomal membrane.
• Cholesterol as a Structural Cofactor: Demonstration that cholesterol binding to TM6SF1 is not merely metabolic but acts as a structural "brace" required for the protein's conformational stability and its ability to engage LAMTOR1.

4. Key Results

• TM6SF1 is Required for mTORC1 Signaling:

  • In TM6SF1 KO cells, mTORC1 signaling is attenuated, evidenced by reduced phosphorylation of p70 S6K and 4E-BP1.
  • Loss of TM6SF1 leads to constitutive nuclear translocation of TFEB (due to lack of mTORC1-mediated phosphorylation), resulting in the upregulation of lysosomal and autophagy genes.
  • Crucially, TM6SF1 deficiency does not alter lysosomal pH or cause lysosomal cholesterol accumulation, indicating a selective role in signaling rather than general lysosomal function or cholesterol trafficking.

• Interaction with the Ragulator Complex:

  • TM6SF1 physically interacts with LAMTOR1. Cross-linking and mutagenesis identified residues Ser89 and Thr90 on LAMTOR1 as critical for this interaction.
  • In TM6SF1 KO cells, LAMTOR1 is mislocalized (diffuse cytosolic/peripheral pattern) rather than being stably anchored to lysosomes, leading to reduced mTORC1 recruitment to the lysosome.

• Structural Insights:

  • TM6SF1 forms a homodimer via interactions between TM7 and TM8 (stabilized by a $\pi$-$\pi$ interaction between Phe273 residues).
  • A cholesterol molecule is bound within a pocket formed by transmembrane helices 1–6. Key residues for binding include Ser150 (hydroxyl group interaction) and aromatic residues (Phe23, Tyr95, Phe154, Tyr177) forming a hydrophobic environment.
  • GC-MS confirmed the presence of cholesterol in purified TM6SF1, and competitive binding assays showed specificity for cholesterol over 25-hydroxycholesterol.

• Cholesterol Binding is Essential for Function:

  • A cholesterol-binding defective mutant (TM6SF1$^{3A}$: N77A/S150A/Y177A) fails to rescue mTORC1 signaling or TFEB cytosolic retention in KO cells, despite localizing correctly to lysosomes.
  • The TM6SF1$^{3A}$ mutant shows a significantly reduced ability to bind the Ragulator complex compared to wild-type.
  • MD simulations reveal that cholesterol binding restricts the conformational flexibility of the cytosolic loop (residues 192–194), locking TM6SF1 in a geometry competent for LAMTOR1 engagement.

5. Significance

This study uncovers a novel, direct mechanistic link between lysosomal cholesterol and growth control:

1. Dual Mechanism of Cholesterol Sensing: The paper distinguishes TM6SF1 from the recently described LYCHOS (GPR155). While LYCHOS senses cholesterol to relieve GATOR1 inhibition (amino acid sufficiency), TM6SF1 acts as a structural scaffold. Cholesterol binding to TM6SF1 stabilizes its dimeric structure, enabling it to anchor the Ragulator complex to the lysosome, which is a prerequisite for mTORC1 docking.
2. Metazoan Specificity: The reliance on TM6SF1 for Ragulator anchoring appears to be a specialized feature of metazoans (absent in yeast), suggesting an evolutionary adaptation for complex nutrient sensing.
3. Therapeutic Implications: Understanding how lysosomal cholesterol regulates mTORC1 via TM6SF1 provides new insights into diseases involving metabolic dysregulation, lysosomal storage disorders, and cancer, where mTOR signaling is often aberrant.

In summary, Hong et al. establish that cholesterol-bound TM6SF1 is a critical structural determinant that stabilizes the Ragulator complex on the lysosomal membrane, thereby enabling the spatial organization required for mTORC1 activation and cellular growth control.