Introduction
Contractile vacuole complexes (CVCs) are a morphologically diverse set
of organelles that are found very broadly throughout eukaryotes, and
particularly in eukaryotic protists. Their recognized role is in
osmoregulation, a critical issue for single-celled organisms inhabiting
freshwater and therefore hyperosmotic environments because constant
passive water uptake across the plasma membrane threatens cellular via
viability, including maintaining physiological cytoplasmic ion
concentrations (Naitoh et al., 1997, Tominaga et al., 1998a, Klauke and
Plattner, 2000, Kissmehl et al., 2002, Tani et al., 2002, Reuter et al.,
2013). Notwithstanding their heterogeneity, CVCs all appear built on
similar organizing principles. They are comprised of one or more large
vacuolar compartments that function as water-storing bladders, that are
ringed by a network of tubular extensions called the spongiome (Allen
and Naitoh, 2002). The existing data are consistent with the general
idea that cytoplasmic water crosses the spongiome membranes via
aquaporins or similar channels and driven by gradients dependent in part
on proton-pumping V+-ATPases. Water in the spongiome
lumen diffuses to swell the connected bladder. At some point, the
bladder membrane fuses with but is not integrated into the plasma
membrane, and the soluble contents are rapidly propelled outside the
cell by dramatic contraction of the bladder. This membrane fusion event
in the Amoebozoan Dictyostelium discoideum is regulated at least
in part by triggered calcium release from the bladder lumen, which acts
to downregulate a CVC-specific Rab (Parkinson et al., 2014). More
generally, ion mobilization from the CVC lumen and/or excretion during
bladder contraction may play homeostatic and/or signalling roles
(Ladenburger et al., 2006, Malchow et al., 2008, Ladenburger et al.,
2009, Patel and Docampo, 2010, Sivaramakrishnan and Fountain, 2012).
Following bladder contraction, the fusion pore to the plasma membrane
reseals and the bladder is ready for re-filling. Though functionally
similar, the precise structures involved in this cycle and their dynamic
interactions may differ in important details between lineages. For
example, in the Ciliate Paramecium the reticular spongiome is comprised
of two distinct zones, and the contractile cycle includes membrane
fission and fusion between the spongiome and bladder (Allen, 2000),
neither of which features in the CVC of the KinetoplastidTrypanosoma cruzi (Jimenez et al., 2022).
CVCs are not found in yeasts or other lineages in which cells secrete
and assemble external walls that can counterbalance osmotic pressures
with physical restraints, nor in multicellular organisms like animals
where the tonicity of the extracellular milieu is under organismal
control. Since the most commonly employed model organisms belong to
these groups, CVCs have been relatively understudied and consequently
many central issues remain unsettled. One largely open question is how
the CVC is integrated with other pathways of eukaryotic membrane
trafficking, including the mechanisms that underlie its maintenance in
vegetative cells and inheritance during cell division. A number of
proteins whose homologs are involved in membrane trafficking in fungi
and animals, have been physically and/or functionally associated with
the CVC in diverse eukaryotic lineages. Early work in D.
discoideum linked both and the clathrin assembly factor AP180 to CVC
function, suggesting a putative connection between endocytic trafficking
and CVC maintenance (Wen et al., 2009, Macro et al., 2012). The CVC is
nonetheless normally isolated from bulk endocytic trafficking, in bothDictyostelium and the Ciliate Tetrahymena thermophila ,
based on studies using endocytic tracers (Gabriel et al., 1999, Cheng et
al., 2023). Whether the same trafficking pathways maintain the CVC in
all lineages remains an open question. A related but more far-reaching
question is whether CVCs emerged independently in different lineages, or
whether they stem from a single ancestral origin. The fact that all
known CVCs rely on vacuolar ATPases might argue for the latter, but
vacuolar ATPases function in a very broad range of compartments in the
endomembrane network (Stevens and Forgac, 1997).
In addition to clathrin and AP180, other endosomal protein homologs were
also shown to be physically and/or functionally associated with the CVC
in Dictyostelium, including AP2, epsin, the endosomal synaptobrevin
homolog Vamp7B, Rabs 11 and 8, and an amphiphysin (I-BAR) family protein
(Harris et al., 2001, Stavrou and O’Halloran, 2006, Wen et al., 2009,
Linkner et al., 2014, Manna et al., 2023, Du et al., 2008, Essid et al.,
2012, Macro et al., 2012). Homologs for a subset of these, including a
Rab11 family member, were subsequently found associated with the CVC inTrypanosoma cruzi (Ulrich et al., 2011). The Ciliates are a
third, very distantly related lineage with prominent CVCs, about which
relatively little is known about membrane trafficking determinants.
Comprehensive localization surveys of SNAREs (in Paramecium
tetraaurelia ) and of Rabs (in T. thermophila ) provided only
ambiguous evidence addressing whether the CVC is an endosome-related
compartment, since in both protein families the most prominent
CVC-localized members did not belong to conserved endosomal subgroups
but instead either belonged to other identifiable subgroups or were
lineage-restricted (Bright et al., 2010, Plattner, 2013, Schonemann et
al., 2013). A possible exception came from the observation that severalT. thermophila Rabs belonging to conserved endocytic/endosomal
subgroups showed secondary localization to the CVC, but with unknown
functional significance (Bright et al., 2010). In contrast, strong
evidence of an endosomal connection in ciliates came from analysis ofT. thermophila Dop1p, a member of the DOPEY family of proteins
that serve endosome-related roles in other lineages. In T.
thermophila , Dop1p localizes strongly to the CVC where it is required
for normal bladder discharge (Cheng et al., 2016, Cheng et al., 2023).
In this manuscript, we present analysis of T. thermophila VPS8D,
a subunit of one of six T. thermophila CORVET (class Ccor e vacuole/e ndosome-t ethering) complexes.
In fungi, animals, and plants, CORVET and the related HOPS
(ho motypic fusion and p rotein s orting)
complexes are key determinants in endo-lysosomal trafficking (van der
Beek et al., 2019). Current data support models in which the
hetero-hexameric complexes tether two vesicle membranes by binding Rab
GTPases in each of the adjoining membranes, and catalyze fusion by
interacting with SNAREs (Baker et al., 2015). While many organisms have
one CORVET and one HOPS, in the lineage leading to T. thermophilaHOPS was lost and CORVET expanded to a family of six complexes, each
with a unique subunit composition and cellular localization (Sparvoli et
al., 2018). One of these complexes, which uniquely contains the subunit
Vpd8D, was localized in an initial survey to the CVC, and subsequently
shown to be present in a punctate distribution at both the bladder and
spongiome, in dynamic equilibrium with a cytosolic pool (Sparvoli et
al., 2020, Cheng et al., 2023). Attempts to study the function of Vpd8Dp
by gene knockout were unsuccessful, since unlike the Vps8 subunits in
other CORVET complexes the VPS8D paralog appeared to be essential
(Sparvoli et al., 2018). Here we have therefore taken a different
approach, using a gene-specific RNA hairpin to partially deplete theVPS8D transcript. The knockdown cells show a variety of
CVC-associated phenotypes. The severity of the defects appears to
correlate with the intensity of the knockdown, and at the extreme
manifests as complete loss of both the bladder and spongiome. Both our
functional and localization data are consistent with the idea that inT. thermophila a ciliate-adapted CORVET complex directs critical
membrane tethering and fusion cycles in the CVC.