An acetylated 120-kDa lysosomal transmembrane protein is absent from mucopolysaccharidosis IIIC fibroblasts: a candidate molecule for MPS IIIC.
ABSTRACT Genetic deficiency of the lysosomal enzyme, acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase), which catalyses the transmembrane acetylation of heparan sulfate results in severe neurodegenerative disease, mucopolysaccharidosis IIIC. N-Acetyltransferase has never been characterized structurally and its gene has never been identified. We combined traditional methods of enzyme purification with organellar proteomics, isolating lysosomal membranes from mouse liver using differential centrifugation and osmolysis, followed by detergent extraction and purification of N-acetyltransferase by liquid chromatography. Partially purified enzyme had a molecular mass of 240 kDa and pI of 7.4 by gel filtration and chromatofocusing. Its specific activity varied with protein concentration typical of oligomeric enzymes or multienzyme complexes. Incubation of N-acetyltransferase with acetyl[14C]CoA in the absence of the acceptor of the acetyl group resulted in radioactive labeling of a 120-kDa polypeptide, suggesting that it represents a subunit containing the enzyme active site. Furthermore, following acetyl[14C]-labeling, the 120-kDa protein was present in the lysosomal membranes purified from the normal human skin fibroblasts but absent in those from the skin fibroblasts of MPS IIIC patients.
- Chemical Reviews 04/2013; 113(4):2733-811. · 41.30 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Heparin acetyl-CoA:alpha-glucosaminide N-acetyltransferase (N-acetyltransferase, EC 22.214.171.124) is an integral lysosomal membrane protein containing 11 transmembrane domains, encoded by the HGSNAT gene. Deficiencies of N-acetyltransferase lead to mucopolysaccharidosis IIIC. We demonstrate that contrary to a previous report, the N-acetyltransferase signal peptide is co-translationally cleaved and that this event is required for its intracellular transport to the lysosome. While we confirm that the N-acetyltransferase precursor polypeptide is processed in the lysosome into a small amino-terminal alpha- and a larger ß- chain, we further characterize this event by identifying the mature amino-terminus of each chain. We also demonstrate this processing step(s) is not, as previously reported, needed to produce a functional transferase, i.e., the precursor is active. We next optimize the biochemical assay procedure so that it remains linear as N-acetyltransferase is purified or protein-extracts containing N-acetyltransferase are diluted, by the inclusion of negatively charged lipids. We then use this assay to demonstrate that the purified single N-acetyltransferase protein is both necessary and sufficient to express transferase activity, and that N-acetyltransferase functions as a monomer. Finally, the kinetic mechanism of action of purified N-acetyltransferase was evaluated and found to be a random sequential mechanism involving the formation of a ternary complex with its two substrates; i.e., N-acetyltransferase does not operate through a ping-pong mechanism as previously reported. We confirm this conclusion by demonstrating experimentally that no acetylated enzyme intermediate is formed during the reaction.PLoS ONE 01/2011; 6(9):e24951. · 3.73 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Rab GTPases regulate vesicle budding, motility, docking, and fusion. In cells, their cycling between active, GTP-bound states and inactive, GDP-bound states is regulated by the action of opposing enzymes called guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). The substrates for most RabGAPs are unknown, and the potential for cross-talk between different membrane trafficking pathways remains uncharted territory. Rab9A and its effectors regulate recycling of mannose 6-phosphate receptors from late endosomes to the trans Golgi network. We show here that RUTBC2 is a TBC domain-containing protein that binds to Rab9A specifically both in vitro and in cultured cells but is not a GAP for Rab9A. Biochemical screening of Rab protein substrates for RUTBC2 revealed highest GAP activity toward Rab34 and Rab36. In cells, membrane-associated RUTBC2 co-localizes with Rab36, and expression of wild type RUTBC2, but not the catalytically inactive, RUTBC2 R829A mutant, decreases the amount of membrane-associated Rab36 protein. These data show that RUTBC2 can act as a Rab36 GAP in cells and suggest that RUTBC2 links Rab9A function to Rab36 function in the endosomal system.Journal of Biological Chemistry 05/2012; 287(27):22740-8. · 4.65 Impact Factor