Four isoforms of the Na؉ /H ؉ exchanger (NHE6 -NHE9) are distributed to intracellular compartments in human cells. They are localized to Golgi and post-Golgi endocytic compartments as follows: mid-to trans-Golgi, NHE8; trans-Golgi network, NHE7; early recycling endosomes, NHE6; and late recycling endosomes, NHE9. No significant localization of these NHEs was observed in lysosomes. The distribution of these NHEs is not discrete in the cells, and there is partial overlap with other isoforms, suggesting that the intracellular localization of the NHEs is established by the balance of transport in and out of the post-Golgi compartments as the dynamic membrane trafficking. The overexpression of NHE isoforms increased the luminal pH of the compartments in which the protein resided from the mildly acidic pH to the cytosolic pH, suggesting that their in vivo function is to regulate the pH and monovalent cation concentration in these organelles. We propose that the specific NHE isoforms contribute to the maintenance of the unique acidic pH values of the Golgi and post-Golgi compartments in the cell.The luminal ionic composition of intracellular compartments differs from the cytoplasm, and each compartment is characterized by a unique, organelle-specific ion concentration. This specific ionic composition is thought to be an important determinant for organelle function and is maintained by the concerted action of ion transport carriers on the membrane (1, 2). Organelles of the secretory and endocytic pathways exhibit differential weak acidity in their lumen with a gradient of pH values decreasing toward the trafficking destination, from ER 1 (pH ϳ7.1) to Golgi (pH ϳ6.2-7.0), trans-Golgi network (TGN) (pH ϳ6.0), and secretory granules (pH ϳ5.0) and from early and late endosomes (pH ϳ6.5) to lysosomes (pH ϳ4.5) (1, 3, 4). This progressive acidification is essential for compartmentalizing cellular events, such as post-translational modifications, sorting of newly synthesized proteins into the secretory pathway, and the degradation or recycling of internalized ligandreceptor complexes and fluid-phase solutes in the endocytic pathway (3, 5). Even pH differences of less than 0.5 between organelles can be essential for the compartmentalizing cellular events (6).The differential ionic milieu of the organelles is maintained by a suite of ion carriers on the membrane, including pumps, channels, and transporters. Luminal acidity is primarily generated by the vacuolar-type H ϩ -translocating ATPase (VATPase) (4, 5).

The detection of microbial pathogens involves the recognition of conserved microbial components by host cell sensors such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs). TLRs are membrane receptors that survey the extracellular environment for microbial infections, whereas NLRs are cytosolic complexes that detect microbial products that reach the cytosol. Upon detection, both sensor classes trigger innate inflammatory responses and allow the engagement of adaptive immunity. Endo-lysosomes are the entry sites for a variety of pathogens, and therefore the sites at which the immune system first senses their presence. Pathogens internalized by endocytosis are well known to activate TLRs 3 and 7-9 that are localized to endocytic compartments and detect ligands present in the endosomal lumen. Internalized pathogens also activate sensors in the cytosol such as NOD1 and NOD2 (ref. 2), indicating that endosomes also provide for the translocation of bacterial components across the endosomal membrane. Despite the fact that NOD2 is well understood to have a key role in regulating innate immune responses and that mutations at the NOD2 locus are a common risk factor in inflammatory bowel disease and possibly other chronic inflammatory states, little is known about how its ligands escape from endosomes. Here we show that two endo-lysosomal peptide transporters, SLC15A3 and SLC15A4, are preferentially expressed by dendritic cells, especially after TLR stimulation. The transporters mediate the egress of bacterially derived components, such as the NOD2 cognate ligand muramyl dipeptide (MDP), and are selectively required for NOD2 responses to endosomally derived MDP. Enhanced expression of the transporters also generates endosomal membrane tubules characteristic of dendritic cells, which further enhanced the NOD2-dependent response to MDP. Finally, sensing required the recruitment of NOD2 and its effector kinase RIPK2 (refs 8, 9) to the endosomal membrane, possibly by forming a complex with SLC15A3 or SLC15A4. Thus, dendritic cell endosomes are specialized platforms for both the lumenal and cytosolic sensing of pathogens.

Human BDCA3 DCs are superior to BDCA1 DCs at antigen cross presentation when delivered to late endosomes and lysosomes but not when delivered to early endosomes.

The VAM2/VPS41 and VAM6/VPS39 were shown to encode hydrophilic proteins of 113 and 123 kDa, respectively. Deletion of the VAM2 and VAM6 functions resulted in accumulation of numerous vacuole-related structures of 200-400 nm in diameter that were much smaller than the normal vacuoles. Loss of functions of Vam2p and Vam6p resulted in inefficient processings of a set of vacuolar proteins, including proteinase A, proteinase B, and carboxypeptidase Y (CPY), and in severely defective maturation of another vacuolar protein, alkaline phosphatase. A part of newly synthesized CPY was missorted to the cell surface in the mutants. Epitope-tagged versions of Vam2p and Vam6p retained their functions, and they were found mostly in sedimentable fractions. The epitope-tagged Vam2p and Vam6p remained in the sedimentable fractions in the presence of Triton X-100, but they were extracted by urea or NaCl. Vam2p and Vam6p were cross-linked by the treatment of a chemical cross-linker. These observations indicated that Vam2p and Vam6p physically interact with each other and exist as components of a large protein complex. Vam6p fused with a green fluorescent protein were highly accumulated in a few specific regions of the vacuolar membranes. Large portions of Vam2p and Vam6p were fractionated into a vacuolar enriched fraction, indicating that they were localized mainly in the vacuolar membranes. These results showed that Vam2p and Vam6p execute their function in the vacuolar assembly as the components of a protein complex reside on the vacuolar membranes.

The vacuole constitutes a large compartment in plant and fungal cells. The VAM3 gene of Saccharomyces cerevisiae encodes a syntaxin-related protein required for vacuolar assembly. An Arabidopsis thaliana cDNA library, designed for expression in S. cerevisiae, was screened for cDNAs able to complement defective vacuolar assembly of the ⌬vam3 mutation. One cDNA, encoding a 33-kDa protein with structural similarities to the other syntaxins, was identified. The product of AtVAM3 (AtVam3p) was expressed in various tissues including roots, leaves, inflorescence stems, flower buds, and young siliques. The AtVAM3 transcripts were abundant in undifferentiated cells in the meristematic region. AtVam3p fractionated predominantly to an 8,000 ؋ g pellet fraction where a vacuolar membrane protein H ؉ -translocating inorganic pyrophosphatase (H ؉ -PPase) also fractionated. Immunoelectron microscopy showed that AtVam3p was localized to restricted regions on the vacuolar membranes. We propose that AtVam3p provides the t-SNARE function in the vacuolar assembly in A. thaliana.The vacuole in plant and fungal cells constitutes a large compartment with various physiological functions (1, 2). The vacuole serves as a storage compartment for various primary and secondary metabolites including amino acids, organic acids, and sugars. It is a lytic compartment as well, sharing common features with lysosomes of mammalian cells. It also functions in homeostatic regulation of cytosolic ions by transporting small molecules across the vacuolar membrane through various transporters. One of the most prominent features of the plant vacuole is its large volume. In fully matured plant cells, it occupies over 90% of cell volume. Individual plants need to expand their stems, leaves, and roots to acquire resources from the environment including light energy, minerals, and water. This expansion in plant body size, however, would be costly if it were accomplished by cell division or by synthesis of cytosolic material. In fact, cell enlargement is mostly attributable to the increase in vacuolar volume. Therefore, the plant vacuole provides a space-filling compartment (3).While the physiological importance of the plant vacuole is clear and many of the molecules that make up this compartment have been well characterized, the molecular mechanisms involved in its assembly are largely unknown. In contrast, vacuolar biogenesis and assembly in the unicellular organism Saccharomyces cerevisiae are fairly well understood. Our previous genetic studies showed that nine VAM genes (for vacuolar morphology) are involved in the assembly of yeast vacuolar compartments (4). We have also shown that the yeast VAM3 gene encodes a member of the syntaxin family (5), the key molecules regulating vesicular transport (6, 7). Syntaxins are membrane receptors on the target membrane (t-SNARE) that interact with the other molecules on the transport vesicles (v-SNARE). Specific interactions between the t-SNARE and v-SNARE molecules ensure fusion of the transport vesicles with their ta...