The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases

Abstract

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs and their precursor, ceramide, are also associated with intracellular organelles and, particularly, mitochondria. GSL biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER), which is transported by controversial mechanisms to the Golgi apparatus, where stepwise addition of monosaccharides on to ceramides takes place. We now report the presence of GSL-biosynthetic enzymes in a subcompartment of the ER previously characterized and termed 'mitochondria-associated membrane' (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide glucosyltransferase (Cer-Glc-T), glucosylceramide galactosyltransferase and sialyltransferase (SAT) activities in the MAM. Estimation of the marker-enzyme activities showed that glycosyltransferase activities could not be ascribed to cross-contamination of MAM by Golgi membranes. Cer-Glc-T was found to have a marked preference for ceramide bearing phytosphingosine as sphingoid base. SAT activities in MAM led to the synthesis of G(M3) ganglioside and small amounts of G(D3). G(M1) was also synthesized along with G(M3) upon incubation of the fraction with exogenous unlabelled G(M3), underlying the presence of other sphingolipid-specific glycosyltransferases in MAM. On the basis of our results, we propose MAM as a privileged compartment in providing GSLs for mitochondria.

Increased localization of APP‐C99 in mitochondria‐associated ER membranes causes mitochondrial dysfunction in Alzheimer disease

“…Previous reports have suggested that MAM is involved in regulating sphingolipid metabolism, affecting mitochondrial activity (Ardail et al , 2003). In fact, mitochondria are reported to contain ceramide, probably generated at MAM (Kogot‐Levin & Saada, 2014).…”

Recycling of Sphingosine Is Regulated by the Concerted Actions of Sphingosine-1-phosphate Phosphohydrolase 1 and Sphingosine Kinase 2

“…A certain pool of ceramide molecules might be selectively translocated to a Golgi sub-fraction for glucosylceramide synthesis by a vesicular and non-CERT pathway (41). Another possibility is that ceramide is converted to hexosylceramides in the ER without inter-organelle transfer, as galactosylceramide synthase is located in the ER, and it has been suggested that de novo synthesis of glucosylceramide takes place not only in the cis-Golgi but also in a sub-region of the ER (42)(43)(44). Interestingly, it has been shown previously that glycosphingolipids are synthesized predominantly from sphingosine salvaged from the lysosomal pathway in slowly dividing cells, whereas in rapidly dividing cells, the need for increased synthesis is met by up-regulation of the de novo pathway (45).…”

Presenilins Are Enriched in Endoplasmic Reticulum Membranes Associated with Mitochondria

“…In some analyses of subcellular fractions, other organelles, including MAM, co-purified with ER, 7 Golgi, 7 or mitochondria. 11 For example, after careful fractionation, sphingolipid-specific glycosyltransferase activity, which previously had been ascribed to the Golgi, was actually found to be in MAM 27 ; in fact, MAM has been described as a pre-Golgi compartment for the secretory pathway. 15 In other cases, the subcellular fractionation separated PS1 into a compartment that was almost certainly MAM, but in the absence of specific MAM markers was either not identified clearly or was identified in nonspecific terms as an ER-related subcompartment.…”

“…16 More than two dozen proteins are concentrated in MAM (see Supplemental Table S1 at http://ajp.amjpathol.org), 15,[17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] including proteins involved in calcium homeostasis (eg, inositol triphosphate receptor isoform 3), in lipid metabolism (eg, fatty acid co-A ligase 4 [FACL4]), in intermediate metabolism (eg, glucose-6-phosphatase), in cholesterol metabolism (eg, acyl-coenzyme A:cholesterol acyltransferase 1), and in the transfer of lipids between the ER and mitochondria. A few nonenzymatic proteins are also concentrated in MAM (see Supplemental Table S1 at http://ajp.amjpathol.org), 15,[17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] suggesting that it is a domain of the ER with specialized functions. Contacts between the two organelles are maintained by MAMassociated proteins, such as phosphofurin acidic cluster sorting protein 2, which controls the apposition of mitochondria with the ER and which appears to stabilize and regulate the interaction of ER and mitochondria.…”