Jan Parolek

Homeostatic pathways maintain the lipid composition of organelle membranes, and mechanistic links between lipid sensing, synthesis, and trafficking are lacking. Acute depletion of cell cholesterol elicits an increase in the rate of very-long-chain (VLC) sphingomyelin synthesis in the Golgi apparatus, thereby promoting cholesterol retention in the plasma membrane. Stable isotope metabolic analyses and lipid trafficking assays showed that the increase in VLC-sphingomyelin results from an increase in the rate of coatomer II–dependent trafficking of VLC-ceramide from the endoplasmic reticulum to the Golgi apparatus. An integral membrane protein of the coatomer II network, cTAGE5, is required for endoplasmic reticulum-to-Golgi trafficking of ceramide and cTAGE5 overexpression caused herniations of the endoplasmic reticulum network that entrapped a synthetic ceramide analog to which cTAGE5 could be photochemically cross-linked. We propose that cTAGE5 is a ceramide sensor for export of VLC-ceramide from the endoplasmic reticulum exit site.

Within cellular membranes, sphingomyelin is associated with cholesterol and this complex facilitates homeostatic regulation of membrane viscosity. Acute cholesterol depletion increases the synthesis of very-long-chain (VLC) sphingomyelin, but a link between lipid sensing and sphingolipid synthesis is lacking. Using sphingolipid metabolic flux analysis, we observed that VLC-ceramide, the precursor to VLC complex sphingolipids that are produced in the Golgi apparatus, was rapidly consumed after cholesterol depletion, while synthesis of long-chain sphingolipids was unaffected. Sphingolipid trafficking assays showed that cholesterol depletion enhances VLC-Ceramide trafficking from the endoplasmic reticulum to the Golgi apparatus. Changes in the sizes of coatomer II ER exit sites were correlated with increased VLC-Ceramide trafficking and concomitant increase in sphingomyelin. Depletion of Sec16A, a component of the COPII network, abolished VLC-SM synthesis. This study reveals ER-to-Golgi trafficking of VLC-Ceramide as a key regulatory node in organelle membrane homeostasis pathways. Summary In cellular membranes, sphingomyelin is associated with cholesterol. Metabolic flux analysis of sphingolipid metabolism showed that synthesis rate of sphingomyelin, but not ceramide, was increased after depletion of cholesterol due increased rate of COPII-dependent ER-to-Golgi transport of ceramide.

Bridge-like lipid transfer protein family member 2 (BLTP2) is an evolutionary conserved protein with unknown function(s). The absence of BLTP2 in Drosophila melanogaster results in impaired cellular secretion and larval death, while in mice ( Mus musculus), it causes preweaning lethality. Structural predictions propose that BLTP2 belongs to the repeating β-groove domain-containing (also called the VPS13) protein family, forming a long tube with a hydrophobic core, suggesting that it operates as a lipid transfer protein (LTP). We establish BLTP2 as a negative regulator of ciliogenesis in RPE-1 cells based on a strong genetic interaction with WDR44, a gene that also suppresses ciliogenesis. Like WDR44, BLTP2 localizes to membrane contact sites involving the endoplasmic reticulum and the tubular endosome network in HeLa cells and that BLTP2 depletion enhanced ciliogenesis in RPE-1 cells grown in serum-containing medium, a condition where ciliogenesis is normally suppressed. This study establishes human BLTP2 as a putative LTP acting between tubular endosomes and ER that regulates primary cilium biogenesis.

Bridge-like lipid transfer protein family member 2 (BLTP2) is an evolutionary conserved protein with unknown function(s). The absence of BLTP2 in Drosophila melanogaster results in impaired cellular secretion and larval death, while in mice ( Mus musculus ), it causes preweaning lethality. Structural predictions propose that BLTP2 belongs to the repeating β-groove domain-containing (also called the VPS13) protein family, forming a long tube with a hydrophobic core, suggesting that it operates as a lipid transfer protein (LTP). We establish BLTP2 as a negative regulator of ciliogenesis in RPE-1 cells based on a strong genetic interaction with WDR44 , a gene that also suppresses ciliogenesis. Like WDR44, BLTP2 localizes to membrane contact sites involving the endoplasmic reticulum and the tubular endosome network in HeLa cells and that BLTP2 depletion enhanced ciliogenesis by serum-fed RPE-1 cells, a condition where ciliogenesis is normally suppressed. This study establishes human BLTP2 as a putative lipid transfer protein acting between tubular endosomes and ER that regulates primary cilium biogenesis. Significance statement: We show the involvement of an ER-localized bridge-like lipid transfer protein, BLTP2, in ciliogenesis and establish that BLTP2 is enriched at organelle-organelle membrane contact sites involving the endoplasmic reticulum (ER) and the tubular endosome network (TEN). These results implicate, for the first time, the involvement of bulk lipid transfer between the ER and TEN in regulating ciliogenesis.