Intracellular trafficking of sphingolipids: Relationship to biosynthesis

The intracellular routes of sphingolipid trafficking are related to the compartmentalized nature of sphingolipid metabolism, with synthesis beginning in the endoplasmic reticulum, continuing in the Golgi apparatus, and degradation occurring mainly in lysosomes. Whereas bulk sphingolipid transport between subcellular organelles occurs primarily via vesicle-mediated pathways, evidence is accumulating that sphingolipids are found in subcellular organelles that are not connected to each other by vesicular flow, implying additional trafficking routes. After discussing how sphingolipids are transported through the secretory pathway, I will review evidence for sphingolipid metabolism in organelles such as the mitochondria, and then discuss how this impacts upon our current understanding of the regulation of intracellular sphingolipid transport.     

Impact of sphingolipids on protein membrane trafficking

Membrane trafficking is essential to maintain the spatiotemporal control of protein and lipid distribution within membrane systems of eukaryotic cells. To achieve their functional destination proteins are sorted and transported into lipid carriers that construct the secretory and endocytic pathways. It is an emerging theme that lipid diversity might exist in part to ensure the homeostasis of these pathways. Sphingolipids, a chemical diverse type of lipids with special physicochemical characteristics have been implicated in the selective transport of proteins. In this review, we will discuss current knowledge about how sphingolipids modulate protein trafficking through the endomembrane systems to guarantee that proteins reach their functional destination and the proposed underlying mechanisms.     

Membrane domains and polarized trafficking of sphingolipids

The plasma membrane of polarized cells consists of distinct domains, the apical and basolateral membrane, that are characterized by a distinct lipid and protein content. Apical protein transport is largely mediated by (glyco)sphingolipid--cholesterol enriched membrane microdomains, so called rafts. In addition changes in the direction of polarized sphingolipid transport appear instrumental in cell polarity development. Knowledge is therefore required of the mechanisms that mediate sphingolipid sorting and the complexity of the trafficking pathways that are involved in polarized transport of both sphingolipids and proteins. Here we summarize specific biophysical properties that underly mechanisms relevant to sphingolipid sorting, cargo recruitment and polarized trafficking, and discuss the central role of a subapical compartment, SAC or common endosome (CE), as a major intracellular site involved in polarized sorting of sphingolipids, and in development and maintenance of membrane polarity.     

细胞内胆固醇运输

作为生物膜的重要成分,细胞内不同膜上胆固醇含量的高低直接影响生物膜的生物物理特性和细胞信号的传递,与细胞正常的生理功能密切相关。外源内吞的胆固醇和内源合成的胆固醇通过囊泡介导和非囊泡介导的胆固醇运输途径在不同细胞膜之间转运,从而维持了不同细胞器上胆固醇的浓度梯度。一系列胆固醇结合和转运蛋白在细胞内胆固醇的运输中发挥了重要作用。本文旨在总结细胞内胆固醇运输途径与参与胆固醇运输的重要分子及相关作用机制。     

Intracellular trafficking of P-glycoprotein

Overexpression of P-glycoprotein (P-gp) is a major cause of multidrug resistance in cancer. P-gp is mainly localized in the plasma membrane and can efflux structurally and chemically unrelated substrates, including anticancer drugs. P-gp is also localized in intracellular compartments, such as endoplasmic reticulum (ER), Golgi, endosomes and lysosomes, and cycles between endosomal compartments and the plasma membrane in a microtubular-actin dependent manner. Intracellular trafficking pathways for P-gp and participation of different Rab proteins depend on cellular polarization and choice of primary culture, cell line or neoplasm. Interruption of P-gp trafficking to the plasma membrane increases intracellular P-gp accumulation and anticancer drug levels, suggesting a potential approach to overcome P-gp-mediated multidrug resistance in cancer.     

Intracellular trafficking of Shiga-toxin-B-subunit-functionalized spherulites

Background information. Spherulites are multi‐lamellar lipidic vesicles that can encapsulate biomolecules and may be used as carriers for drug delivery. STxB (Shiga toxin B‐subunit) is known to bind the glycosphingolipid Gb3 (globotriaosyl ceramide), which is overexpressed by various human tumours. After Gb3 binding, the toxin enters the cytoplasm via the retrograde route, bypassing the degrading environment of the late endosomes/lysosomes. STxB is non‐toxic and has been identified as a promising tool for drug delivery. So far, applications have relied on direct coupling with therapeutic agents. In the present study, we have investigated the functionalization of spherulites by STxB and the intracellular trafficking of these structures. Results. We demonstrate that STxB‐spherulites (ST×B‐functionalized spherulites) are internalized into HeLa cells in a receptor‐dependent manner. The intracellular distribution was studied by confocal microscopy for lipids, ligand and content. We observed an early separation between spherulites and STxB, leading to a late endosomal/lysosomal localization of lipids and content, whereas STxB remained partially at the plasma membrane. Conclusions. Although recognition of Gb3 is the cause of their specific adhesion to cell membranes, STxB‐spherulites do not follow the retrograde transport route. Our results strongly suggest that STxB‐spherulites are, at least in part, disrupted at the plasma membrane, leading to lipid and content targeting to the classical endocytic pathway. We discuss how these findings influence the development of innovative delivery strategies.     

Intracellular trafficking of Parachlamydia acanthamoebae

Parachlamydia acanthamoebae is an obligate intracellular bacterium that naturally infects free-living amoebae. It is a potential agent of pneumonia that resists destruction by human macrophages. However, the strategy used by this Chlamydia-like organism in order to resist to macrophage destruction is unknown. We analysed the intracellular trafficking of P. acanthamoebae within monocyte-derived macrophages. Infected cells were immunolabelled for the bacteria and for various intracellular compartments by using specific antibodies. We analysed the bacteria colocalization with the different subcellular compartments by using epifluorescence and confocal microscopy. Bacterial replication took place 4-6 h post infection within acidic vacuoles. At that time, P. acanthamoebae colocalized with Lamp-1, a membrane marker of late endosomal and lysosomal compartments. A transient accumulation of EEA1 15 min post infection, and of rab7 and the mannose 6-phosphate receptor 30 min post infection confirmed that P. acanthamoebae traffic through the endocytic pathway. The acquisition of Lamp-1 was not different after infection with living and heat-inactivated bacteria. However, 24.5% and 79.5% of living and heat-inactivated P. acanthamoebae, respectively, colocalized with the vacuolar proton ATPase. Moreover, P. acanthamoebae did not colocalized with cathepsin D, a lysosomal hydrolase, suggesting that P. acanthamoebae interferes with maturation of its vacuole. Thus, P. acanthamoebae survives to destruction by human macrophages probably by controlling the vacuole biogenesis.     

Intracellular trafficking of TRP channels

Thirteen years ago, it was suggested that exocytotic insertion of store-operated channels into the plasma membrane lead to increased Ca(2+) entry in non-excitable cells upon G protein-coupled or tyrosine kinase receptor stimulation. Since the discovery of the TRP channel superfamily and their involvement in receptor-induced Ca(2+) entry, many studies have shown that different members of the TRP superfamily translocate into the plasma membrane upon stimulation. While the exact molecular mechanism by which TRP channels insert into the plasma membrane is unknown, TRP-binding proteins have been shown to directly regulate this trafficking. This review summarizes recent advances related to the mechanism of TRP channel trafficking, focusing on the role of TRP-binding proteins.     

Intracellular iron trafficking: role of cytosolic ligands

Iron acquired by cells is delivered to mitochondria for metabolic processing via pathways comprising undefined chemical forms. In order to assess cytosolic factors that affect those iron delivery pathways, we relied on microscopy and flow-cytometry for monitoring iron traffic in: (a) K562 erythroleukemia cells labeled with fluorescent metal-sensors targeted to either cytosol or mitochondria and responsive to changes in labile iron and (b) permeabilized cells that retained metabolically active mitochondria accessible to test substrates. Iron supplied to intact cells as transferrin-Fe(III) or Fe(II)-salts evoked concurrent metal ingress to cytosol and mitochondria. With either supplementation modality, iron ingress into cytosol was mostly absorbed by preloaded chelators, but ingress into mitochondria was fully inhibited only by some chelators, indicating different cytosol-to-mitochondria delivery mechanisms. Iron ingress into cytosol or mitochondria were essentially unaffected by depletion of cytosolic iron ligands like glutathione or the hypothesized 2,5 dihydroxybenzoate (2,5-DHBA) siderophore/chaperone. These ligands also failed to affect mitochondrial iron ingress in permeabilized K562 cells suspended in cytosol-simulating medium. In such medium, mitochondrial iron uptake was >6-eightfold higher for Fe(II) versus Fe(III), showed saturable properties and submicromolar K(1/2) corresponding to cytosolic labile iron levels. When measured in iron(II)-containing media, ligands like AMP, ADP or ATP, did not affect mitochondrial iron uptake whereas in iron(III)-containing media ADP and ATP reduced it and AMP stimulated it. Thus, cytosolic iron forms demonstrably contribute to mitochondrial iron delivery, are apparently not associated with DHBA analogs or glutathione but rather with resident components of the cytosolic labile iron pool.     

Intracellular trafficking of thyroid peroxidase to the cell surface

For thyroid hormone synthesis, thyroid peroxidase (TPO) molecules must be transported from the endoplasmic reticulum via the Golgi complex to be delivered at the cell surface to catalyze iodination of secreted thyroglobulin. Like other glycoproteins, TPO molecules in transit to the cell surface have the potential to acquire endoglycosidase H resistance as a consequence of Golgi-based modification of their N-linked carbohydrates, and measurement of the intracellular distribution of TPO has often relied on this assumption. To examine TPO surface distribution in thyrocyte cell lines, we prepared new antibodies against rat TPO. Antibody reactivity was first established upon expression of recombinant rat (r) TPO in 293 cells, which were heterogeneous for surface expression as determined by flow cytometry. By cell fractionation, surface rTPO fractionated distinctly from internal pools of TPO (that co-fractionate with calnexin), yet surface TPO molecules remained endoglycosidase H (endo H)-sensitive. Although the FRTL5 (and PC Cl3) rat thyrocyte cell line also exhibits almost no endo H-resistant TPO, much of the endogenous rTPO is localized to the cell surface by immunofluorescence. Similar results were obtained by fractionation of FRTL5 cell membranes on sucrose gradients. We conclude that in FRTL5 cells, a large fraction of rTPO is delivered to the plasma membrane yet does not acquire Golgi-type processing of its N-glycans. Rat and mouse thyroid tissue TPO also shows little or no endo H resistance, although cell fractionation still needs to be optimized for these tissues.     

Intracellular trafficking in Plasmodium-infected erythrocytes

The past few years have witnessed considerable progress in molecular and biochemical studies of intracellular trafficking in malaria-infected red cells. Highlights include the identification of solute channels in the vacuolar membrane and the red blood cell membrane, a tubovesicular membrane network that delivers exogenous nutrients and drugs to the parasite, and parasite gene families that mediate adherence to endothelial cells and red cells.     

Intracellular trafficking in the trypanosomatids

Trypanosomes are members of the kinetoplastida, a group of divergent protozoan parasites responsible for considerable morbidity and mortality worldwide. These organisms have highly complex life cycles requiring modification of their cell surface together with engagement of immune evasion systems to effect survival; both processes intimately involve the membrane trafficking system. The completion of three trypanosomatid and several additional protist genomes in the last few years is providing an exciting opportunity to evaluate, at the molecular level, the evolution and diversity of membrane trafficking across deep evolutionary time as well as to analyse in unprecedented detail the membrane trafficking systems of trypanosomes.     

Intracellular trafficking of cell surface sialoglycoconjugates

Recent reports have suggested that the majority of the molecular traffic through the Golgi apparatus is comprised of recycling, rather than newly synthesized, molecules. To evaluate the importance of this recycling pathway in greater detail, we examined the internalization and recycling of cell surface glycoproteins on EL-4 cells, a murine T-cell lymphoma, using sialic acids as covalent markers. Sialic acids were removed from the surface of living cells by exhaustive treatment with Vibrio cholerae sialidase at 4 degrees C and shown to be derived primarily from glycoproteins (93%), with only a small amount from glycolipids (7%). Cells were recultured at 37 degrees C over time and monitored for the resialylation of the cell surface using a sensitive high pressure liquid chromatography adaptation of the thiobarbituric acid assay for sialic acids. The return of sialic acid to the cell surface was found to be contingent upon de novo protein synthesis indicating that the bulk of plasma membrane sialoglycoconjugates do not recycle to an endogenous sialyltransferase-containing compartment for oligosaccharide reprocessing. Identical results were found for K562 cells, a human erythroleukemia cell line. The movement of specific glycoproteins was followed using the enzyme rat liver alpha 2-6Gal beta 1-4GlcNAc sialyltransferase together with CMP-[3H]NeuAc as an impermeant probe of the cell surface. Surface sialoglycoproteins were internalized slowly, a process unaffected by cycloheximide treatment. Only a few of these internalized glycoproteins were found to return to a trans-Golgi compartment followed by recycling to the cell surface. Taken together, these data indicate that the majority of replacement of sialic acids on the cell surface is due to de novo synthesis of glycoproteins and that only a small number of glycoproteins recycle through a trans-Golgi compartment.