ABSTRACT
The cytoplasmic regions of neurotransmitter transporters play an important role in their trafficking. This process is, to a high extent, tuned by calcium and calcium binding proteins, but the exact molecular connection are still not fully understood. In this work we found that the C-terminal region of the mouse glycine transporter GlyT1b is able to specifically interact with calmodulin in the presence of calcium. We found that several GlyT1 C-terminal mutations, including those in the ER retention signal, either eliminate or increase calmodulin interaction in vitro. In tissue-culture-expressed GlyT1 at least two of these mutations altered the sensitivity of GlyT1 surface expression and glycine uptake to calmodulin antagonists. These results suggest the possible involvement of calmodulin or calmodulin-like interactions in the regulation of GlyT1C-mediated transporter trafficking.
Subject(s)
Calcium/metabolism , Calmodulin/metabolism , Glycine Plasma Membrane Transport Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line , Glycine/metabolism , Glycine Plasma Membrane Transport Proteins/chemistry , Mice , Protein TransportABSTRACT
Previously, we found that the C-terminus of the glycine transporter GlyT1 loses the most of its epitopes during pathological calcium increase in rodent synaptosomes but that the more internal epitopes are spared. We also found that epitope immunoreactivity likely decreases via both phosphorylation and calpain-mediated proteolysis. Here we show that the predicted phosphomimetic mutation S605D fully blocks the adjacent (T602/T603) internal calpain cleavage in the mouse GlyT1b C-terminal fusion protein, but that the neutral S605A mutation does not. Consistent with this, the phophomimetic mutation, but not the neutral mutation, significantly protected the internal GlyT1 C-terminal antiGlyT1C603-626 epitopes against calpain when introduced into tissue culture expressed GlyT1b. Because similar effects can be obtained using phosphatase inhibitors, it may be that phosphorylation of S605 protects the GlyT1 C-terminal sequences from calpain cleavage in vivo.
Subject(s)
Calpain/metabolism , Glycine Plasma Membrane Transport Proteins/metabolism , Animals , Cell Line, Tumor , Glycine Plasma Membrane Transport Proteins/chemistry , Glycine Plasma Membrane Transport Proteins/genetics , Mice , Mutation , PhosphorylationABSTRACT
We previously demonstrated that glycine transporters GlyT1 and GlyT2 are differentially affected by toxic benzophenanthridine alkaloids. Using a combination of homology modeling, knowledge of the sensitivity of sanguinarine to sulfhydryl reagents and site directed mutagenesis we show here that the increased sensitivity of human GlyT1c to sanguinarine is abolished by the mutation of only cysteine 475. Inhibition requires the membrane permeable alkaloid alkanolamine, which is consistent with the intracellular location of the targeted cysteine.
Subject(s)
Anti-Infective Agents/toxicity , Benzophenanthridines/toxicity , Glycine Plasma Membrane Transport Proteins/drug effects , Isoquinolines/toxicity , Mutation , Cell Membrane Permeability , Cysteine/chemistry , Cysteine/genetics , Glycine Plasma Membrane Transport Proteins/chemistry , Glycine Plasma Membrane Transport Proteins/metabolism , HEK293 Cells , Humans , Mutagenesis, Site-Directed , Protein Structure, TertiaryABSTRACT
Comparative analysis of the telomeres of distantly related species has proven to be helpful for identifying novel components involved in telomere maintenance. We therefore initiated such a study in the nonconventional yeast Yarrowia lipolytica. Its genome encodes only a small fraction of the proteins that are typically associated with telomeres in other yeast models, indicating that its telomeres may employ noncanonical means for their stabilization and maintenance. In this report, we have measured the size of the telomeric fragments in wild-type strains, and characterized the catalytic subunit of telomerase (YlEst2p). In silico analysis of the YlEst2 amino acid sequence revealed the presence of domains typical for telomerase reverse transcriptases. Disruption of YlEST2 is not lethal, but results in retarded growth accompanied by a rapid loss of the telomeric sequences. This phenotype is associated with structural changes at the chromosomal ends in the ΔYlest2 mutants, likely the circularization of all six chromosomes. An apparent absence of several typical telomere-associated factors, as well as the presence of an efficient means of telomerase-independent telomere maintenance, qualify Y. lipolytica as an attractive model for the study of telomere maintenance mechanisms and a promising source of novel players in telomere dynamics.
