Macrophages are important targets for HIV-1 infection and harbor the virions in an as yet unidentified organelle. To determine the location of HIV-1 in these cells, an extensive analysis of primary human macrophages infected in vitro with HIV-1 was carried out by immuno-electron microscopy. Virus particles were found to accumulate in intracellular multivesicular compartments which were enriched in major histocompatibility complex class II molecules and CD63. These features are characteristics of major histocompatibility complex class II compartments where maturing class II molecules acquire their peptide cargo. The membrane-delimited, electron-dense virus particles of 100-110 nm diameter labeled strongly for HIV-1 p24 antigen, major histocompatibility complex class II molecules, CD63 and, to a lesser extent for HIV-1 gp120 envelope protein and Lamp 1. Our data suggest that virus particles may access the lumen of the major histocompatibility complex class II compartment by budding from the limiting membrane, thus acquiring proteins of this membrane such as class II and CD63. Viral assembly and budding would therefore occur in macrophages by a process similar to the formation of the internal vesicles in multivesicular bodies and at the same location. This could account for the particular content in lipids and proteins previously found in the membrane wrapping HIV particles. Our observations also suggest direct fusion of the virus containing major histocompatibility complex class II compartment with the plasma membrane, leading to massive release of viral particles into the extracellular medium.

Mutations in the aquaporin-2 ( AQP2 ) water channel gene cause autosomal recessive nephrogenic diabetes insipidus (NDI). Here we report the first patient with an autosomal dominant form of NDI, which is caused by a G866A transition in the AQP2 gene of one allele, resulting in a E258K substitution in the C-tail of AQP2. To define the molecular cause of NDI in this patient, AQP2-E258K was studied in Xenopus oocytes. In contrast to wild-type AQP2, AQP2-E258K conferred a small increase in water permeability, caused by a reduced expression at the plasma membrane. Coexpression of wild-type AQP2 with AQP2-E258K, but not with an AQP2 mutant in recessive NDI (AQP2-R187C), revealed a dominant-negative effect on the water permeability conferred by wild-type AQP2. The physiologically important phosphorylation of S256 by protein kinase A was not affected by the E258K mutation. Immunoblot and microscopic analyses revealed that AQP2-E258K was, in contrast to AQP2 mutants in recessive NDI, not retarded in the endoplasmic reticulum, but retained in the Golgi compartment. Since AQPs are thought to tetramerize, the retention of AQP2-E258K together with wild-type AQP2 in mixed tetramers in the Golgi compartment is a likely explanation for the dominant inheritance of NDI in this patient. ( J. Clin. Invest. 1998. 102:57-66.)

UDP-galactose:ceramide galactosyltransferase (CGalT) transfers UDP-galactose to ceramide to form the glycosphingolipid galactosylceramide. Galactosylceramide is the major constituent of myelin and is also highly enriched in many epithelial cells, where it is thought to play an important role in lipid and protein sorting. Although the biochemical pathways of glycosphingolipid biosynthesis are relatively well understood, the localization of the enzymes involved in these processes has remained controversial. We here have raised antibodies against CGalT and shown by immunocytochemistry on ultrathin cryosections that the enzyme is localized to the endoplasmic reticulum and nuclear envelope but not to the Golgi apparatus or the plasma membrane. In pulse-chase experiments, we have observed that newly synthesized CGalT remains sensitive to endoglycosidase H, confirming the results of the morphological localization experiments. In protease protection assays, we show that the largest part of the protein, including the amino terminus, is oriented toward the lumen of the endoplasmic reticulum. CGalT enzyme activity required import of UDP-galactose into the lumen of the endoplasmic reticulum by a UDP-galactose translocator that is present in the Golgi apparatus of CHO cells but absent in CHOlec8 cells. Finally, we show that CGalT activity previously observed in Golgi membrane fractions in vitro, in the absence of UDP-glucose, is caused by UDPglucose:ceramide glucosyltransferase. Therefore all galactosylceramide synthesis occurs by CGalT in vivo in the lumen of the endoplasmic reticulum.

SummaryIn human B lymphoblastoid cell lines, the majority of major histocompatibility complex (MHC) class II heterodimers are located on the cell surface and in endocytic compartments, while invariant chain (Ii)-associated class II molecules represent biosynthetic intermediates which are present mostly in the endoplasmic reticulum and Golgi complex. To investigate the origin of the MHC class II-positive compartments and their relation to early endosomes, the intracellular distribution of MHC class II molecules and Ii in relation to endocytic tracers was studied in human lymphoblastoid B cells by immunoelectronmicroscopy on ultrathin cryosections. Cross-linking of surface immunoglobulins, followed by a brief period of internalization of the immune complexes, did not alter the intracellular distribution of MHC class II molecules. While early endosomes were abundantly labeled for the cross-linked immunoglobulins, < 1% of total MHC class II molecules were detectable in early endosomes. MHC class II-and Ii-positive structures associated with the trans-Golgi network can be reached by endocytosed bovine serum albumin (BSA)-gold conjugates after 30 min of internalization. Prolonged exposure to BSA-gold allowed visualization of later endocytic compartments, in which a progressive loss of Ii was observed: first the lumenal portion, and then the cytoplasmic portion of Ii escaped detection, culminating in the formation of MHC class II-positive compartments (MIIC) devoid of Ii. The loss of Ii also correlated with a transition from a multivesicular to a multilaminar, electron-dense MIIC. The intracellular compartments in which class II molecules reside (MIIC) are therefore a heterogeneous set of structures, part of the later aspects of the endocytic pathway.

Abstract. Misfolded membrane proteins are rapidly degraded, often shortly after their synthesis and insertion in the endoplasmic reticulum (ER), but the exact location and mechanisms of breakdown remain unclear. We have exploited the requirement of MHC class I molecules for peptide to achieve their correct conformation: peptide can be withheld by introducing a null mutation for the MHC-encoded peptide transporter, TAP. By withholding TAP-dependent peptides, the vast majority of newly synthesized class I molecules fails to leave the endoplasmic reticulum and is degraded. We used mice transgenic for HLA-B27 on a TAPl-deficient background to allow visualization by immunoelectron microscopy of misfolded HLA-B27 molecules in thymic epithelial cells. In such HLA transgenic animals, the TAP mutation can be considered a genetic switch that allows control over the extent of folding of the protein of interest, HLA-B27, while the rate of synthesis of the constituent subunits remains unaltered.,In TAPl-deficient, HLA-B27 transgenic animals, HLA-B27 molecules fail to assemble correctly, and do not undergo carbohydrate modifications associated with the Golgi apparatus, such as conversion to Endoglycosidase H resistance, and acquisition of sialic acids. We show that such molecules accumulate in an expanded network of tubular and fenestrated membranes. This compartment has its counterpart in normal thymic epithelial cells, and is identified as an ER-Golgi intermediate. We detect the presence of ubiquitin and ubiquitin-conjugating enzymes in association with this compartment, suggesting a nonlysosomal mode of degradation of its contents.