Characterization of the MAL2-positive compartment in oligodendrocytes.

Oligodendrocytes (OLs), the myelin-producing cells of the central nervous system, segregate different surface subdomains at the plasma membrane as do other differentiated cells such as polarized epithelia and neurons. To generate the complex membrane system that characterizes myelinating OLs, large amounts of membrane proteins and lipids need to be synthesized and correctly targeted. In polarized epithelia, a considerable fraction of apical proteins are transported by an indirect pathway involving a detour to the basolateral membrane before being internalized and transported across the cell to the apical membrane by a process known as transcytosis. The apical recycling endosome (ARE) or its equivalent, the subapical compartment (SAC), of hepatocytes is an intracellular trafficking station involved in the transcytotic pathway. MAL2, an essential component of the machinery for basolateral-to-apical transcytosis, is an ARE/SAC resident protein. Here, we show that, after differentiation, murine oligodendrocyte precursor and human oligodendroglioma derived cell lines, Oli-neu and HOG, respectively, up-regulate the expression of MAL2 and accumulate it in an intracellular compartment, exhibiting a peri-centrosomal localization. In these oligodendrocytic cell lines, this compartment shares some of the main features of the ARE/SAC, such as colocalization with Rab11a, sensitivity to disruption of the microtubule cytoskeleton with nocodazole, and lack of internalized transferrin. Therefore, we suggest that the MAL2-positive compartment in oligodendrocytic cells could be a structure analogous to the ARE/SAC and might have an important role in the sorting of proteins and lipids for myelin assembly during oligodendrocyte differentiation.

Dynamics of MAL2 during glycosylphosphatidylinositol-anchored protein transcytotic transport to the apical surface of hepatoma HepG2 cells.

Delivery of glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface takes place by transcytosis in hepatocytes and also probably in epithelial Madin-Darby canine cells. The integral protein MAL2 was demonstrated to be essential for basolateral-to-apical transcytosis in hepatoma HepG2 cells. Reduction of endogenous MAL2 levels impedes cargo delivery to the apical membrane, but, paradoxically, cargo does not accumulate in the subapical compartment where MAL2 predominantly resides but in distant endosome elements. To understand how transcytosis can be apparently mediated at a distance, we have analyzed the dynamics of machinery and cargo by live-cell imaging of MAL2 and transcytosing CD59, a GPI-anchored protein, in HepG2 cells. MAL2 was revealed as being a highly dynamic protein. Soon after basolateral endocytosis of CD59, a fraction of MAL2 redistributed into peripheral vesicular clusters that concentrated CD59 and that were accessible to transferrin (Tf) receptor, a basolateral recycling protein. Following Tf receptor segregation, the clusters fused in a MAL2(+)globular structure and moved toward the apical surface for CD59 delivery. All these processes were impaired in cells with reduced MAL2 content. Other GPI-anchored proteins examined behave similarly. As MAL2 is expressed by many types of epithelia, the sorting events described herein are probably of quite general utility.

Caveolin-1 and MAL are located on prostasomes secreted by the prostate cancer PC-3 cell line.

MAL, BENE and MAL2 are raft-associated integral membrane proteins of the MAL family of proteins involved in membrane trafficking processes. We show here that the human prostate carcinoma PC-3 cell line expresses the transcripts for the three proteins simultaneously. MAL, BENE and MAL2 co-fractionated with caveolin-1 in the raft fraction of PC-3 cells, and immunofluorescence analysis showed colocalization of these proteins with caveolin-1 in a multivesicular intracellular compartment. Markers of the Golgi apparatus, early and recycling endosomes and lipid droplets were excluded from this compartment. Prostate epithelial cells contain vesicular organelles enriched in raft components named prostasomes that are secreted in the prostate fluid. Interestingly, the prostasome fraction isolated from the culture supernatant of PC-3 cells consisted mainly of 30-130 nm cup-shaped vesicles that were positive for MAL, caveolin-1 and CD59, a glycosylphosphatidylinositol-anchored protein previously found in prostasomes. CD63, an integral membrane protein found in multivesicular bodies/lysosomes and secretory granules was also found in PC-3 cell-derived prostasomes. Prostasome secretion was not inhibited by brefeldin A, a compound that blocks the conventional secretory pathway. However, wortmannin, an inhibitor of phosphatidylinositol-3 kinase, reduced the secretion of prostasomes in PC-3 cells. Our results suggest that MAL family proteins are associated with caveolin-1 in a multivesicular compartment that may be involved in prostasomal secretion in PC-3 cells.

Expression of MAL2, an integral protein component of the machinery for basolateral-to-apical transcytosis, in human epithelia.

MAL2, an integral membrane protein of the MAL family, is an essential component of the machinery necessary for the indirect transcytotic route of apical transport in human hepatoma HepG2 cells. To characterize the range of human epithelia that use MAL2-mediated pathways of transport, we carried out an immunohistochemical survey of normal tissues using a monoclonal antibody specific to the MAL2 protein. MAL2 expression was detected in specific types of normal epithelial cells throughout the respiratory system, the gastrointestinal and genitourinary tracts, in exocrine and endocrine glands, and in hepatocytes. Many different types of specialized secretory cells, either organized in discrete clusters (e.g., endocrine cells in the pancreas) or in endocrine glands (e.g., prostate), were also positive for MAL2. In addition to epithelial cells, peripheral neurons, mast cells, and dendritic cells were found to express MAL2. For comparison with normal epithelial tissue, different types of renal carcinoma were also analyzed, revealing alterations in MAL2 expression/distribution dependent on the particular histological type of the tumor. Our results allow the prediction of the existence of MAL2-based trafficking pathways in specific cell types and suggest applications of the anti-MAL2 antibody for the characterization of neoplastic tissue.

Expression and distribution of MAL2, an essential element of the machinery for basolateral-to-apical transcytosis, in human thyroid epithelial cells.

Polarized transport of newly synthesized proteins to the apical surface of epithelial cells takes place by a direct pathway from the Golgi or by an indirect route involving the delivery of the protein to the basolateral surface, followed by its endocytosis and transport across the cell. The indirect pathway, named transcytosis, is also used to translocate external material across the cell. MAL, a raft-associated integral membrane protein required for the direct apical route, is known to be expressed in the thyroid epithelium. MAL2, a member of the MAL protein family, has been recently identified as an essential component of the machinery for the transcytotic route in human hepatoma cells. Herein, we have investigated the expression and distribution of MAL2 in the human thyroid. MAL2 mRNA species were detected in the thyroid. Immunohistochemical analysis of thyroid follicles indicated that, in contrast to MAL, which predominantly distributed to the Golgi region, MAL2 distributed to the apical membrane. Biochemical analysis in primary thyrocyte cultures indicated that MAL2 exclusively resides in raft membranes. Confocal immunofluorescence analysis of thyrocyte cultures revealed that MAL2 predominantly localized in a subapical endosome compartment that was positive for Rab11a. Alterations in MAL2 expression, distribution, and appearance were found in specific types of follicular cell-derived carcinomas. Although the role of MAL2 has not been directly addressed in this study, the simultaneous expression of MAL and MAL2 suggests that traffic to the apical membrane in thyrocytes may rely on MAL for the direct route and on MAL2 for the transcytotic pathway.

MAL2, a novel raft protein of the MAL family, is an essential component of the machinery for transcytosis in hepatoma HepG2 cells.

Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi (e.g., epithelial MDCK cells) as an indirect route for targeting proteins to the apical surface. The raft-associated MAL protein is an essential element of the machinery for the direct route in MDCK cells. Herein, we present the functional characterization of MAL2, a member of the MAL protein family, in polarized HepG2 cells. MAL2 resided selectively in rafts and is predominantly distributed in a compartment localized beneath the subapical F-actin cytoskeleton. MAL2 greatly colocalized in subapical endosome structures with transcytosing molecules en route to the apical surface. Depletion of endogenous MAL2 drastically blocked transcytotic transport of exogenous polymeric immunoglobulin receptor and endogenous glycosylphosphatidylinositol-anchored protein CD59 to the apical membrane. MAL2 depletion did not affect the internalization of these molecules but produced their accumulation in perinuclear endosome elements that were accessible to transferrin. Normal transcytosis persisted in cells that expressed exogenous MAL2 designed to resist the depletion treatment. MAL2 is therefore essential for transcytosis in HepG2 cells.