The objective of this research proposal is to obtain a molecular understanding of the
phosphomannosyl targeting system which functions in the delivery of newly synthesized acid
hydrolases to lysosomes. Genetic defects in this intracellular protein transport pathway give
rise to severe lysosomal storage diseases, illustrating the importance of this system. A key step
in this pathway is the selective phosphorylation of mannose residues on the high mannose
glycans of the acid hydrolases by UDP-GlcNAc:lysosomal enzymes N-acetylgucosamine-1-
phosphotransferase (Ptase). This cis-Golgi transferase is a type III transmembrane a2ß2¿2
hexameric protein encoded by two genes. We established that the two Notch repeat modules
and the DNA methyltransferase-associated protein (DMAP) interaction domain of the a subunit
mediate the specific recognition of the common protein determinant of acid hydrolases. Specific
Aim 1 is directed toward defining the role of each domain in this process. Aim 2 focuses on the
role of the N-terminal cytoplasmic tail of Ptase in maintaining proper Golgi localization. This aim
is derived from our finding that three ML III patient missense mutations in the cytoplasmic N-tail
of the a-subunit result in mislocalization of the mutant Ptase to the endosome/lysosome system.
Our hypothesis is that these mutations prevent recycling of this Golgi enzyme due to a failure to
be incorporated into COPI vesicles, a required step for the recycling process. In support of this,
our preliminary findings show direct binding of the WT N-tail, but not the mutant peptides, to the
d and ¿ subunits of COPI. The goal is to understand these interactions at the molecular level.
Aim 3 is to determine the generality of the direct interaction of the cytoplasmic N-tails of the
large family of Golgi type II transmembrane glycosyltransferases with the COPI subunits.
Strong evidence exists that the glycosyltransferases recycle from late to early Golgi cisternae or
the ER via COPI vesicles, but how the transferases are packaged into the vesicles is poorly
understood. This aim has the potential to establish a new paradigm for this process.