Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules

Abstract

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal ␤/␥-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, ⌬GRT1 ⌬GRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in ⌬GRT1 ⌬GRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from ⌬GRT1 ⌬GRT2 cells appear less adhesive than those from the wild type.

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

“…Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. discover that a set of genes encoding mucocyst cargo proteins, a VPS10 family receptor involved in cargo sorting, and several putative proteases were all transcriptionally coregulated (16,27,34). Among those proteases is a gene for cathepsin 4 (CTH4), whose expression pattern was strikingly similar to that of several other mucocyst-associated genes (Fig.…”

“…Dibucaine and alcian blue stimulation of exocytosis was performed as described previously (27,34). Importantly, cells were washed and suspended in fresh medium immediately prior to stimulation.…”

Conservation and Innovation in Tetrahymena Membrane Traffic: Proteins, Lipids, and Compartments

“…In T. thermophila these vesicles are called mucocysts, and they have been explored as a system for understanding biosynthetic mechanisms that are still rather poorly understood in animals, but which appear to differ from canonical mechanisms involved in vesicle formation(Tooze et al, 2001; Turkewitz, 2004). Mucocysts contain two major families of soluble (i.e., not membrane-bound) proteins, which are coordinately expressed(Rahaman et al, 2009). The first, whose corresponding genes were called GRL for granule lattice, are required to form a protein crystal that comprises the bulk of the dense core(Cowan et al, 2005).…”

“…Two members of this family have been investigated and neither is proteolytically processed(Bowman et al, 2005a; Haddad et al, 2002). In addition, none of the genes in this family that have been disrupted is essential for core assembly, but the double disruption of two related genes subtly changed the properties of the mucocyst core following its exocytic release, so apparently the proteins in this family are playing distinct roles from the Grls(Rahaman et al, 2009). Only one protein in the non-Grl family has been localized and was found, remarkably, to be highly concentrated at the end of the secretory granule where it docks at the plasma membrane, prior to exocytosis(Bowman et al, 2005a).…”

Constitutive Secretion in Tetrahymena thermophila

“…Because strain SB281 does not produce mucocysts, GRL protein secretion cannot result from a spontaneous background of mucocyst discharge. Recently, an unanticipated complexity of mucocyst cargo trafficking steps has been recognized that may provide the cellular pathway(s) for shunting of mucocyst cargo to constitutive secretion (29). We suggest that, in all of the strains, GRL proteins precluded from mucocyst condensation may be constitutively secreted in soluble form.…”