Proteolytic Processing and Ca²⁺-binding Activity of Dense-Core Vesicle Polypeptides inTetrahymena

Abstract

Formation and discharge of dense-core secretory vesicles depend on controlled rearrangement of the core proteins during their assembly and dispersal. The ciliate Tetrahymena thermophila offers a simple system in which the mechanisms may be studied. Here we show that most of the core consists of a set of polypeptides derived proteolytically from five precursors. These share little overall amino acid identity but are nonetheless predicted to have structural similarity. In addition, sites of proteolytic processing are notably conserved and suggest that specific endoproteases as well as carboxypeptidase are involved in core maturation. In vitro binding studies and sequence analysis suggest that the polypeptides bind calcium in vivo. Core assembly and postexocytic dispersal are compartment-specific events. Two likely regulatory factors are proteolytic processing and exposure to calcium. We asked whether these might directly influence the conformations of core proteins. Results using an in vitro chymotrypsin accessibility assay suggest that these factors can induce sequential structural rearrangements. Such progressive changes in polypeptide folding may underlie the mechanisms of assembly and of rapid postexocytic release. The parallels between dense-core vesicles in different systems suggest that similar mechanisms are widespread in this class of organelles. INTRODUCTIONDense-core secretory vesicles, also called secretory granules, are specialized vesicles with a condensed protein core (Halban and Irminger, 1994). The vesicle core can be described as a dynamic aggregate. First, it is assembled within the secretory pathway from previously soluble proteins. Conversion of a set of soluble proteins to a temporarily insoluble form can serve diverse functions, including their efficient storage in a state that imposes a reduced osmotic burden. Secondly, when an appropriate exocytic stimulus triggers the fusion of the vesicle membrane with the plasma membrane, the dissemination of the secreted contents involves re-or dis-assembly of the core. Granule core dynamics exemplify the potential for compartmentspecific regulation of protein interactions in the secretory pathway (Arvan and Castle, 1992), and offer the opportunity to investigate the underlying mechanisms.Interactions among proteins in transit may be influenced by their microenvironments, as for example gradients of pH and calcium concentration within and between secretory compartments. The potential importance of such modulation is not limited to granule assembly. For example, the assembly of T-cell receptor subunits may be controlled by their calcium-specific binding to BiP (Suzuki et al., 1991), and ligand-binding by the ER retrieval receptor may be regulated by the pH difference between the ER and the Golgi (Wilson et al., 1993). For proteins forming the dense cores of neuroendocrine granules, a combination of these factors in the TGN and newly formed secretory vesicles have been implicated in the induction of aggregation/ condensation (references in Natori and Hut...

Identification and Characterization of a Novel Secretory Granule Calcium-binding Protein from the Early Branching Eukaryote Giardia lamblia

“…Regulated secretory proteins form a vesicle core that was described as a dynamic aggregate (45,46). After their synthesis in the ER, regulated secretory proteins are sorted and segregated into secretory granules as soluble proteins; inside the vesicles, these proteins are converted to a temporarily insoluble form and finally reassembled after exocytosis, adopting a most stable structure.…”

“…In Tetrahymena thermophila and Paramecium tetraurelia, for example, the vesicle core synthesis occurs by assembly of soluble proteins into an insoluble, well organized lattice (46,57). It was proposed that in both microorganisms, this process involves binding of extracellular ionic calcium to regulated secretory proteins (46,58).…”

Calcium in ciliated protozoa: Sources, regulation, and calcium-regulated cell functions

“…In tndl cells, 45Ca2+ binding of several bands is reduced in gel overlays, the precise genetic basis remaining to be established. In Tetrahymena, this Ca 2 + sensitivity develops during proteolytic processing of mucocyst content (Verbsky and Turkewitz, 1998). Any Ca 2 +-dependent biosynthetic processes during maturation thus can be exeuted without internal "explosion."…”

Secretion of Polypeptide Crystals from Tetrahymena thermophila Secretory Organelles (Mucocysts) Depends on Processing by a Cysteine Cathepsin, Cth4p

“…During exocytosis, the secretory proteins are rapidly extruded, due to the fact that the crystalline lattices expand rapidly and directionally. Thus, the function of secretory organelles in ciliates depends upon the assembly of protein crystals that can undergo spring-like elongation (20,21).The most abundant proteins stored in and released from Tetrahymena mucocysts belong to two families, called GRT (for granule tip) and GRL (for granule lattice) (22,23). Studies of the Grl proteins, and the related P. tetraurelia tmp proteins, have demonstrated that extensive proteolytic processing occurs during mucocyst and trichocyst synthesis (20,(24)(25)(26).…”

“…Thus, the function of secretory organelles in ciliates depends upon the assembly of protein crystals that can undergo spring-like elongation (20,21).…”