Sequential cleavage of the proteins encoded by HNOT/ALG3, the human counterpart of the Drosophila NOT and yeast ALG3 gene, results in products acting in distinct cellular compartments.

This study provides first insights into the biosynthesis, structure, biochemistry and complex processing of the proteins encoded by hNOT/ALG3, the human counterpart of the Drosophila Neighbour of TID (NOT) and the yeast asparagine linked glycosylation 3 gene (ALG3), which encodes a mannosyltransferase. Unambiguous evidence that both the fly and human proteins act as mannosyltransferases has not been provided yet. Previously, we showed that hNOT/ALG3 encodes two alternatively spliced main transcripts, hNOT-1/ALG3-1 and hNOT-4/ALG3-4, and their 15 truncated derivatives that lack diverse sets of exons and/or carry point mutations that result in premature termination codons. Here we show that the truncated transcripts are not translated. The two main forms hNOT-1/ALG3-1 and -4, distinguishable by alternative exon 1, encode full-length precursors that undergo a complex posttranslational processing. To specifically detect the two full-length hNOT/ALG3 proteins and their distinct derivatives and to examine their expression profiles and cellular location we generated polyclonal antibodies against diverse parts of the putative full-length proteins. We provide experimental evidence for the N-glycosylation of the two precursors. This modification seems to be a prerequisite for their sequential cleavage resulting in derivatives destined to distinct cellular compartments and links them with the N-glycosylation machinery not as its functional component but as molecules functionally dependent on its action. We present the expression profiles and subcellular location of the two full-length proteins, their N-glycosylated forms and distinct cleavage products. Furthermore, using diverse bioinformatics tools, we characterize the properties and predict the 2D and 3D structure of the two proteins and, for comparative purposes, of their Drosophila counterpart.

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