Isolation and characterization of an apically sorted 41-kDa protein from the midgut of tobacco hornworm (Manduca sexta)

Immunocytochemical localization and sorting properties of a newly purified 41-kDa protein (MsM41) were investigated in an insect, the tobacco hornworm Manduca sexta. The protein purified from midgut homogenates of feeding fif th-stadium larvae was found exclusively in this tissue on Western blots. Pr esence of MsM41 protein was indicated in both anterior and posterior region s of the midgut during the whole fifth stadium. However, in the posterior r egion an additional 39-kDa protein was also detected during the feeding per iod of the last larval stage. Upon light-microscopic examination immunoreac tivity was localized in the columnar cells, while the goblet, endocrine and regenerative cells remained unlabeled. Distribution of the label during th e feeding period was different in the anterior and posterior regions. In th e anterior region immunoreactivity was localized only to the brush border m embrane of columnar cells, while in the posterior region some cytoplasmic s tructures identified as large trans-Golgi vesicles, endoplasmic reticulum a nd small secretory vesicles were also labeled. Large, apical extrusions rem ained immunonegative. In vitro translation confirmed that our protein was e xpressed only in the posterior region of the midgut. The primary translatio n product was a 39-kDa protein. Putative post-translational modifications y ielded the 41-kDa form, which was then secreted apically. Its presence in t he region of the anterior part microvilli was probably due to the countercu rrent flux of the ectoperitrophic fluid.