A facilitated diffusion for glycerol is present in human erythrocytes. Glycerol transporters identified to date belong to the Major Intrinsic Protein (MIP) family of integral membrane proteins, and one of them, aquaporin-3 (AQP3), has been characterized in mammals. Using an antibody raised against a peptide corresponding to the rat AQP3 carboxyl terminus, we examined the presence of AQP3 in normal and Colton-null (aquaporin-1 (AQP1)-deficient) human erythrocytes. Three immunoreactive bands were detected on immunoblots of both normal and Colton-null red cells, very similar to the bands revealed in rat kidney, a material in which AQP3 has been extensively studied. By immunofluorescence, anti-AQP3 antibodies stained the plasma membranes of both normal and Colton-null erythrocytes. Glycerol transport was measured on intact erythrocytes by stopped-flow light scattering and on one-step pink ghosts by a rapid filtration technique. Glycerol permeability values, similar in both cell types, suggest that AQP1 does not represent the major path for glycerol movement across red blood cell membranes. Furthermore, pharmacological studies showed that Colton-null red cells remain sensitive to water and glycerol flux inhibitors, supporting the idea that another proteinaceous path, probably AQP3, mediates most of the glycerol movements across red cell membranes and represents part of the residual water transport activity found in AQP1-deficient red cells.Human erythrocytes are highly permeable to water, urea, and glycerol (1-3). The existence of membrane proteins that facilitate water and solute movements in these cells has been postulated, but most of these proteins remain to be characterized. In 1992, Agre and co-workers identified a very abundant protein of the human red blood cell as the first water-selective channel that was named aquaporin-1 (AQP1) 1 (4). Colton-null erythrocytes, lacking AQP1, exhibit a reduced osmotic water permeability (5). However, these cells are found to be residually permeable to water, suggesting the presence of additional, non-AQP1, water channels.The existence of a protein-mediated glycerol transport in erythrocytes has relied mostly on pharmacological evidence. In particular, inhibition of glycerol transport by sulfhydryl reagents, phloretin, and copper ions has been reported (1,3,6). Yet, the identity of the erythrocyte glycerol carrier remains unknown. The glycerol facilitators identified to date all belong to the Major Intrinsic Protein (MIP) family (7). They have been characterized in several organisms: GlpF, a bacterial glycerol permease facilitator (8); Fps1p, a yeast glycerol exporter (9); aquaporin-3 (AQP3), initially characterized in mammalian kidney (10 -12); and AQP7, recently identified in rat testis (13). Compared with other mammalian aquaporins, which are selective mostly for water, AQP3 is moderately permeable to water, but highly permeable to glycerol and possibly to urea (11,12,14). AQP3 expression has been reported in several mammalian tissues, including kidney, intestine, stomach...

Class E vacuolar protein-sorting (Vps) proteins were first described in yeast as being involved in receptormediated endocytosis and multivesicular body formation. Inactivation by RNA interference of the class E VPS genes of the nematode Caenorhabditis elegans revealed heterogeneous phenotypes. We have further characterized the role of the essential gene Cevps-27, ortholog of human hepatocyte growth factor-regulated tyrosine kinase substrate, during the development of C. elegans. Use of green fluorescent protein fusion constructs and antibody staining revealed that Cevps-27 localizes to endosomal membranes. It is widely expressed but enriched in epithelial cells. Cevps-27 mutants presented enlarged endosomal structures and an accumulation of autophagic vesicles as revealed by electron microscopy and the analysis of the autophagic marker LGG-1. Cevps-27 animals arrested at L2-L3 molt with an inability to degrade their old cuticle. This molting phenotype was more severe when Cevps-27 worms were grown on suboptimal concentrations of cholesterol. Furthermore, defective endocytic trafficking of the low-density lipoprotein receptor-related protein 1 (LRP-1) was also observed in Cevps-27 mutants. These results indicate that CeVPS-27 is required for endosomal and autophagic pathways in C. elegans and plays a crucial role in the control of molting through LRP-1 internalization and cholesterol traffic.

AQP3 is a water and glycerol channel present on human erythrocytes and in various tissues. By protein and molecular biology analysis, two unrelated probands who developed alloantibodies to the high frequency antigen GIL were found to be AQP3-deficient. The defect is caused by homozygous mutation affecting the 5 donor splice site of intron 5 of the AQP3 gene. This mutation causes the skipping of exon 5 and generates a frameshift and premature stop codon. Functional studies by 90°l ight scattering using a stopped-flow spectrometer revealed the absence of facilitated glycerol transport across red cell membranes from the probands, but the water and urea transports were normal. Expression studies into COS-7 cells followed by flow cytometry analysis showed that only cells transfected with AQP3 cDNA strongly reacted with anti-GIL antibodies. These findings represent the first reported cases of AQP3 deficiency in humans and provide the molecular basis of a new blood group system, GIL, encoded by the AQP3 protein.Integral membrane proteins that facilitate the transport of water or/and solutes belong to the major intrinsic protein (MIP) 1 family and are involved in many physiological processes and the pathophysiology of several clinical disorders (1-4). They are divided into three subgroups according to their sequences and function properties: (i) the aquaporins (AQP), permeable only to water; (ii) the glycerol facilitators, permeable only to glycerol (not present in mammals); and (iii) the aquaglyceroporins, which present a mixed selectivity. AQP1, the archetype of the MIP family selectively permeable to water (5), is present in various tissues including red blood cell (RBC) membranes from which it was first purified (6).The high glycerol permeability of human RBCs is due to aquaglyceroporin AQP3 (7,8), which is moderately permeable to water, highly permeable to glycerol, and to a lesser extent permeable to urea (9 -11). AQP3 is present in rat RBCs but absent from mouse RBCs (8). It is encoded by a single-copy gene composed of six exons distributed over 6 kilobases of DNA located on human chromosome 9p13 (12). The predicted protein of 292 residues is organized into six bilayer-spanning domains with the NH 2 -and COOH termini located intracellularly (13). On human RBCs, AQP3 presumably is glycosylated on all subunits at Asn-141, the putative N-glycosylation site located in loop "c" at the external face of the cell membrane (14).So far, AQP1 and AQP3 are the only two known proteins of the aquaporin family identified in human RBCs. As AQP1 express Colton blood group antigens (15, 16), the question was raised as to whether AQP3 might also be encoded by a blood group gene. We reasoned that if AQP3 carries a blood group specificity, the corresponding antigen should be of high frequency because AQP3 is a common protein of human RBCs. Accordingly, we performed an immunostaining analysis of human RBCs proteins from patients who had developed alloantibodies against high frequency antigens that caused delayed or severe hemolytic ...