ABSTRACT
The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.
Subject(s)
Endosomes/metabolism , Protein Transport/physiology , rab4 GTP-Binding Proteins/metabolism , Cell Compartmentation/drug effects , Cell Compartmentation/physiology , Endocytosis/drug effects , Epidermal Growth Factor/metabolism , Gene Expression , Genes, Dominant , HeLa Cells , Humans , Iodine Radioisotopes , Ligands , Lipoproteins, LDL/metabolism , Lipoproteins, LDL/pharmacokinetics , Microtubules/metabolism , Mutagenesis, Site-Directed , Protein Transport/drug effects , Receptors, Transferrin/metabolism , Transfection , Transferrin/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , rab GTP-Binding Proteins/pharmacology , rab4 GTP-Binding Proteins/genetics , rab4 GTP-Binding Proteins/pharmacology , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/metabolism , rab5 GTP-Binding Proteins/pharmacology , rab7 GTP-Binding ProteinsABSTRACT
Rab4 belongs to the Rab family of small GTPases involved in the regulation of intracellular transport, and has been localized to early endosomes. We have employed the yeast two-hybrid system to identify proteins that specifically interact with Rab4AQ67L, a GTPase-deficient mutant form of Rab4A. Screening a mouse embryo cDNA library identified a clone (M449) that interacted with Rab4A in a nucleotide-dependent fashion. Data base searches identified this clone as the mouse cytoplasmic dynein light intermediate chain-1 (LIC-1). Based on this finding, the full-length equivalent human cytoplasmic dynein LIC-1 was isolated by PCR. When Rab4A was overexpressed together with either M449 or dynein LIC-1 in HeLa cells, the proteins were found to colocalize in the perinuclear region. We characterize the localization of both overexpressed human dynein LIC-1 and the endogenous protein with respect to microtubules and show that it concentrates to the microtubule-organizing center and mitotic spindle. Additionally, GFPRab4A endosomes localize to microtubules and are redistributed by nocodazole treatment. This is the first described interaction between cytoplasmic dynein, a retrograde motor protein, and a Rab protein.
