Lipid juggling: Any1 scrambles membranes for endosome biogenesis

To selectively transport membrane proteins and lipids into lysosomes for degradation, endosomes undergo dynamic remodeling of their lipid bilayer (1). This is particularly notable during the biogenesis of multivesicular bodies (MVBs), which consist of a limiting membrane and intraluminal vesicles (ILVs). How lipid homeostasis is maintained during MVB biogenesis was not well understood.

Now, Jieqiong Gao and colleagues reveal that Any1 (SLC66A2/PQLC1 in humans) functions as an endosome and Golgi-resident phospholipid scramblase (Fig. 1) (2). Alphafold modeling of Any1 (and its human orthologs SLC66A2) with molecular dynamics simulations and in vitro experiments show robust lipid scrambling activity. This lipid scramblase is essential for MVB formation. On endosomes, Any1 collaborates with the bridge-like lipid transfer protein Vps13 (3) to facilitate lipid transfer from lipid droplets (LDs) and/or the ER.

Vps13-mediated unidirectional lipid transfer at ER–endosome contact sites had been implied earlier in MVB biogenesis (4). Since Vps13 does not penetrate the bilayer and only accesses the cytosolic leaflets of membranes, its lipid transfer capacity will partially deplete the cytosolic leaflet of the donor membrane and selectively expand the cytosolic leaflet of the acceptor membranes over time. Hence, it was unclear how lipid homeostasis was maintained in endosomal membranes. With the identification of Any1 as the missing scramblase on endosomes, Gao et al. provide the first mechanistic insight into how the transferred lipids are vertically distributed between the inner and outer leaflets of endosomal membranes. It appears that Any1 could function conceptually similar to Atg9 on autophagosomes, which cooperates with another bridge-like lipid transfer protein (Atg2) during phagophore expansion (5). It remains unclear whether Any1 and Vps13 interact directly, or if their function is coordinated by molecular mechanisms that do not rely on direct protein–protein interaction.

How would lipid transfer and scrambling support MVB biogenesis? MVBs are specialized endosomes that package cargo such as ubiquitinated membrane proteins and lipids for degradation in ILVs (1). ILV biogenesis is catalyzed by the endosomal sorting complexes required for transport (ESCRT). The formation of each ILV requires five ESCRT sub-complexes. The molecular architecture of these multi-subunit protein complexes, as well as how they interact with one another, with endosomal lipids, and with ubiquitinated membrane proteins, is understood in atomic detail (6). ESCRT-0, -I, -II capture and concentrate ubiquitinated cargo and thus define a membrane area on endosomes that can mature into an ILV. These early ESCRT complexes trigger the assembly of ESCRT-III subunits and the AAA-ATPase Vps4 into dynamic heteropolymeric filaments. The ESCRT-III/Vps4 assemblies drive the membrane remodeling and scission processes that bud ILVs into the lumen of endosomes. Repetition of this process fills the lumen of endosomes with ILVs, leading to maturation of MVBs (7). The fusion of mature MVBs with lysosomes releases ILVs into the lumen of lysosomes, where they are degraded with their cargo.

While significant progress had been made in understanding the molecular machineries governing MVB biogenesis, one critical aspect remained poorly understood: How do MVBs coordinate membrane homeostasis of the limiting membrane with ILV biogenesis? After all, the formation of each ILV removes lipids and proteins, thereby shrinking the surface area of the endosome, while simultaneously expanding its volume. Hence, without additional lipid supply, an MVB would reach maximal filling prematurely, resulting in a maximal surface:volume ratio and high membrane tension. This would prevent the formation of additional ILVs. In such a scenario, ubiquitinated membrane proteins might remain on the endosomal surface, prohibiting MVB maturation. Conversely, low endosomal membrane tension stimulates ESCRT-III assembly (8).

Vps13-directed lipid transfer and Any1-mediated scrambling is critical to support complete MVB maturation, especially during a stage of MVB maturation, where fusion with early endosomes no longer occurs. Of note, Vps13-mediated lipid supply alone is insufficient for MVB biogenesis and requires Any1-dependent lipid scrambling. Gao et al. (2) show that deletion of Any1 results in a significant reduction in the number of MVBs, indicating a delay in MVB biogenesis. Consistently, the sorting of MVB cargo, such as the methionine permease Mup1, is severely impaired in Any1-deficient cells.

One intriguing finding of this study is the potential involvement of LDs in endosome biogenesis. LDs store triglycerides and cholesterol esters and have been shown to form membrane contact sites (MCSs) with various organelles, including the ER and mitochondria. The functional significance of LD–endosome interactions remains poorly understood.

Using live-cell imaging, Gao et al. (2) detect transient LD–endosome proximity during endocytosis of the methionine transporter Mup1, which is dependent on Vps13. To better characterize if LDs are within 10–40 nm of endosomes to establish MCSs, high-pressure freezing and transmission electron microscopy were used. LD–endosome MCSs are occasionally detected, likely due to their transient nature. Yet, in ESCRT-deficient cells, where MVB biogenesis is disrupted and multi-lamellar class E compartments (with a large surface) form instead, LD class E MCSs are frequently detected.

Together, these findings support a tripartite MCS involving LDs, endosomes, and the ER, ensuring efficient and partially redundant Vps13- and Any1-mediated lipid mobilization for endosome maturation. The concept of tripartite MCSs has previously been described for mitochondria, the ER, and vacuoles (vCLAMP) (9, 10).

If lipid transfer indeed occurs from LDs, neutral lipids and sterol esters may also be transferred to endosomes, where they play a hitherto uncharacterized role during MVB biogenesis, or they are metabolized (en route) into phospholipids and sterols. Alternatively, but equally interesting: Vps13 and Any1 might help to extract lipids from maturing endosomes toward LDs, perhaps to get rid of excess neutral lipids and/or cholesterol.

While a function of Any1 during MVB biogenesis is now elucidated, it remains unclear if and how Any1-mediated lipid scrambling contributes to Golgi function, or if the scramblase activity of Any1 is spatially regulated, being most active on endosomes and less so on other organelles.

In conclusion, this study establishes Any1 as a phospholipid scramblase that is required for efficient MVB biogenesis, positioning it as a key component of the endocytic pathway.

Several key questions can be addressed in the future:

• (1)

  Which lipids are transferred between LDs, the ER, and endosomes, and in which direction?

• (2)

  Is Any1 activity spatially regulated, and does it directly interact with Vps13?

• (3)

  How are the transient MCSs between endosomes and LDs regulated, and which factors are additionally involved in transient LD–endosome MCS formation?

Given that human SLC66A2/PQLC1 shares structural similarities with Any1, this study paves the way for studying the role of endosomes and Golgi-resident scramblases in lipid metabolism and trafficking in human health and disease.