LIMITED PROTEOLYSIS OF (1,3)-BETA-GLUCAN (CALLOSE) SYNTHASE FROM BETA-VULGARIS L - TOPOLOGY OF PROTEASE-SENSITIVE SITES AND POLYPEPTIDE IDENTIFICATION USING PRONASE-E

Plasma membrane (PM) vesicles of defined sidedness were obtained from Beta vulgaris L. and subjected to limited proteolysis to investigate t he topology and subunit composition of UDP-glucose: (1,3)-beta-glucan (callose) synthase (CalS). Latency experiments demonstrated that prote ase-sensitive sites on the CalS complex are located primarily at the c ytoplasmic face of the PM, with little or no CalS inactivation occurri ng as the result of proteolysis at the apoplastic face. In the PM-boun d form, CalS activity was resistant to inactivation by Pronase E, howe ver at least four polypeptides previously implicated as possible CalS components (92, 83, 57 and 43 kDa) were extensively hydrolyzed. Polype ptides of 31, 29 and 27 kDa resisted Pronase E hydrolysis and were als o enriched in CalS fractions purified by glycerol gradient centrifugat ion and product entrapment. In contrast to PM-bound CalS, purified Cal S was rapidly hydrolyzed by Pronase E, indicating that most Pronase E- sensitive sites are deeply embedded within the PM. This study provides direct biochemical evidence that hydrophobic integral membrane protei ns oriented primarily towards the cytoplasmic face of the PM are impor tant for callose biosynthesis in Beta. Furthermore, these results form the basis of a biochemically derived working model largely consistent with morphologically derived models proposed for intramembrane PM-bou nd, microfibril-synthesizing complexes in higher plants.