Our objective is to understand the biochemistry and cell biology of glycoprotein processing and secretion. Yeast, like higher mammalian cells, secrete glycoproteins by transport through a compartmentalized exocytotic pathway. Glc3Man9GlcNAc2, transferred to nascent peptides in the rough endoplasmic reticulum (RER), is processed in this compartment by removal of Glc3 and a specific Man to generate a homogeneous Man8GlcNAc2 """"""""processing intermediate,"""""""" which is elongated by Man transferases in subcombparments of the Golgi complex to a family of Man9-14GlcNAc2 species. Structurally, Man8-14GlcNAc2 from secreted invertase are homogeneous isomers defining the pathway of mannan synthesis on this enzyme. These studies will be extended by determining to what extent the invertase pathway reflects global glycan processing in yeast in general, and the processing of carboxypeptidase Y (CPY), a non-secreted glycoprotein targetted to the vacuole (the yeast lysosome counterpart), in particular. Invertase and CPY will be isolated from wild-type, alg3 and sec mutants which arrest exocytosis early in the pathway. Their endoglycosidase-released oligosaccharides will be purified and structures determined by high-field 1H NMR spectrsocopy, confirmed as necessary by fast atom bombardment/mass spectroscopy, exoglycosidase digestions and chemical means. Processing pathways will be compared to determine whether the sec mutants operationally define new compartments between the RER (sec18) and the trans Golgi (sec7) where glycan processing is complete. Immunoelectron microscopy will be used to localize invertase and CPY in normal and sec yeast to see whether they functionally traverse the secretory pathway together. The RER Alpha1,2-mannosidase will be purified and antibodies raised for immunoscreening yeast transformed with yeast genomic DNA to identify, isolate and clone this gene for studies on the in vivo significance of processing through Man9GlcNAc2. That a """"""""correct"""""""" carbohydrate configuration is indispensable for the proper function of certain glycoproteins is shown by the diverse array of human diseases resulting from the failure to correctly synthesize, process, or degrade their glycan moieties. Clearly, glycoproteins are one of the essential building blocks of integrated biological systems, and fundamental knowledge of how these products are made and function within the organism is essential to providing a rational basis for detecting, understanding and curing identifiable disease states.
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