Abstract
The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α’’. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.
Highlights
Membrane trafficking is an essential process responsible for maintaining the structure, composition and functions of eukaryotic cells [1]
The Trf1 mutant was previously shown to be severely defective in the transfer of Lucifer yellow [10], and this was subsequently shown to be corrected by overexpression of CK2a’’
The lowest degree of dye coupling was manifested in the Trf3 mutant (Fig. 1B). These results demonstrate that gap junction-mediated intercellular communication is defective in each Trf mutant
Summary
Membrane trafficking is an essential process responsible for maintaining the structure, composition and functions of eukaryotic cells [1]. There are two major membrane trafficking routes, endocytic and exocytic, that govern regulated transport between the plasma membrane, Golgi apparatus, endoplasmic reticulum (ER), endosomes and lysosomes [2]. The endocytic pathway is used for the internalization of macromolecules such as signaling receptors from the plasma membrane. Internalized molecules are sorted to early endosomes and, either directed to late endosomes and subsequently to lysosomes for degradation, or recycled back to the cell surface directly, or via recycling endosomes [3,4,5]. The exocytic pathway, on the other hand delivers newly synthesized proteins from the ER, through the Golgi apparatus to the plasma membrane [6]. Each step of membrane trafficking - cargo selection, vesicle formation, vesicle movement along cytoskeletal elements, tethering and fusion with target membrane - is stringently controlled [7]. Of key importance is the superfamily of RAB GTPases that ensure efficient transport of cargo to the appropriate destination [2,7,8]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have