In higher eukaryotic cells, specialized lysosomes with unique molecular properties are developed to carry out unique physiological processes, and they are collectively referred to as lysosome-related organelles (LROs; Dell’Angelica 2000 ; Raposo and Marks, 2002 ; Shiflett 2002 ). undesirable proteins, glycans, lipids, and additional molecules (Kornfeld and Mellman, 1989 ; Mellman, 1996 ; Gruenberg, 2001 ; Jerome and Yancey, 2003 ). Problems in lysosomal function have been shown to be associated with varied human diseases, collectively referred to as lysosomal storage diseases (Neufeld, 1991 ; Walkley, 1998 ; Desnick and Schuchman, 2002 ; Germain, 2002 ; Ward 2002 ). In normal cultured cells, lysosomes are distributed throughout in the cell with higher concentrations in the perinuclear region. In higher eukaryotic cells, specialised lysosomes with unique molecular properties are developed to carry out unique physiological processes, and they are collectively referred to as lysosome-related organelles (LROs; Dell’Angelica 2000 ; Raposo and Marks, 2002 ; Shiflett 2002 ). For example, melanosomes generated by melanocytes are important for pigmentation and protective part of the organism (Marks and Seabra, 2001 ). The class II compartments in antigen-presenting cells are LROs that are specifically involved in processing antigens into antigenic peptides for loading onto the class II major histocompatibility molecules, a key event in our immune defense (Pieters, 1997 ; Honey and Rudensky, 2003 ). Lytic granules in natural killer cells and cytotoxic T lymphocytes, platelet-dense granules in platelets, and granules in basophils and neutrophils are additional examples of LROs (Dell’Angelica 2000 ; Rendu and Brohard-Bohn, 2001 ; Djeu 2002 ). The morphology and distribution of lysosomes and/or LROs are intimately linked to their functions and the underlying molecular mechanisms are being actively investigated (Barbosa 1996 ; Nagle 1996 ; Oh 1996 , 2000 ; Gardner 1997 ; Faigle 1998 ; Dell’Angelica 2000 ; Raposo 2001 ; Shiflett 2002 ; BQCA Ward 2002 ). Several proteins have been recently shown to possess the house of clustering lysosomes in the perinuclear central region, including Vam6p, Rab7, Rab34, and RILP (Bucci 2000 ; Caplan 2001 ; Cantalupo 2001 ; Jordens 2001 ; Wang and Hong, 2002 ). Rab7 and Rab34 are both capable of interacting with RILP, and this connection is definitely limited to the wild-type and GTP-restricted mutant, but not the GDP-restricted mutant of these two Rabs (Cantalupo 2001 ; Wang and Hong, 2002 ). The effect of Rab34 on lysosomal morphology and distribution is absolutely dependent on its connection with RILP. Although the possibility that Rab7 could regulate lysosomes inside a RILP-independent manner through additional effectors has not been formally excluded, its ability to interact with RILP may be similarly important for Rab7 to regulate lysosomal morphology. In this case, the function of Rab7, Rab34, and RILP may converge on a common pathway. Vam6p seems to regulate lysosomes inside a Rab7-self-employed manner, suggesting that it either functions downstream of or functions in parallel with Rab7 (Caplan 2001 ). With this statement, we recognized two novel proteins (RLP1 and RLP2) that are structurally BQCA related to RILP via the presence of two homologous areas. It was then functionally shown that RILP, but not RLP1 and RLP2, has a specific part in regulating lysosomal morphology and distribution. A 62-residue region unique to the 401-residue RILP was exposed to be necessary for RILP’s function to regulate lysosome as well as adequate to confer a similar property, when transferred, to RLP1. A strong correlation was observed between the ability to regulate lysosomal morphology and connection with Rab7 (or simultaneous connection with both Rab7 and Rab34), suggesting this connection could be the mechanism underlying RILP’s ability to regulate lysosomes. This summary was further sustained from the observation the 62-residue region of RILP can confer RLP1 the ability to interact efficiently with Rab7 and Rab34. MATERIALS AND METHODS Cells and Antibodies Normal rat kidney (NRK) and Hela cells were cultivated in DMEM supplemented with 10% fetal bovine serum (Existence Systems, Ann Arbor, MI) inside a 5% CO2 incubator at 37C. The mAb (Mab) against myc tag, rat light1 and a Golgi SNARE GS28 were described earlier (Subramaniam 1996 ; NARG1L Wang and Hong, 2002 ). Mab against TGN38 was a gift from G. Banting (University or college of Bristol, UK; Luzio 1990 ). Mab against transferrin receptor and EEA1 were from BD Transduction Laboratories (Lexington, KY). Polyclonal antibody against myc tag was from Upstate Biotechnology (Lake Placid, NY). FITC- BQCA and rhodamine-conjugated secondary antibodies were.