Cells known to spew secretory protein granules had been helpful in deciphering and defining the secretory pathway. But what happened when secretion systems were turned off? Lysosomes were known to degrade foreign proteins taken up by cells and even autodigest intracellular membranous structures—but what was the fate of excess endogenous protein?
To ask that question, Smith and Farquhar (1966) needed a secretion system that could be manipulated in the lab (and without the benefit of today's inducible gene expression systems). Lactating rats provided prolactin-secreting pituitary cells that fit the bill as “it was easy to cut off secretion by removing the suckling babies and then ask, how would the cells adapt?” says Marilyn Farquhar (University of California, San Diego, CA).
The cells, says Farquhar, were “devoted to pushing out prolactin,” at least until the babies were removed. At that point the duo brought in the new and powerful technique of enzyme histochemistry to localize lytic activity (Miller and Palade, 1964), which could “bridge the gap between [fractionation] biochemistry and EM.”
The traditional assumption was that cells would simply store excess secretory granules until they were needed again. So the researchers were surprised to observe that immature granules fused with multivesicular bodies (MVBs) and mature granules fused with “dense bodies” or lysosomes. Previous work suggested that MVBs were intermediates in lysosomal degradation of endocytosed proteins in renal and nerve cells (Farquhar and Palade, 1962; Rosenbluth and Wissig, 1964). Furthermore, Smith and Farquhar's acid phosphatase tests showed lytic activity in the MVBs that contained granules. This led the authors to postulate that MVBs “can take up and digest proteins and are transformed in the process into” lysosomes. The idea was very close to the concept of maturation followed by fusion with lysosomes as suggested 30 years later by Futter et al. (1996).
Because the prolactin granules retained a characteristic size and density for some time after fusion, the paper demonstrated for the first time that endogenous proteins could also enter the lysosomal degradation pathway. The paper also noted that excess ER and ribosomes, ramped up for prolactin production, were down-regulated by autophagic structures that also converged on the lysosomal pathway.
Farquhar notes that one of the prolific namers of the times, Christian de Duve, dubbed the observed secretory granule down-regulation “crinophagy.” And she points out that we have yet to answer a key question of the crinophagy pathway: “How does that [granule] membrane get changed in such a way that it goes to the lysosome instead of the plasma membrane?”