VPS8D knockdown affects features of both the bladder and
spongiome
Conventional CORVET in S. cerevisiae acts as a determinant in the
maturation of endosomal compartments (Peplowska et al., 2007). To ask
whether knockdown of T. thermophila VPS8D led to changes
in the CVC, we generated cells expressing the VPS8D hairpin in
combination with endogenously tagged copies of either Dop1p, which most
strongly labels the bladder, or tagged Vma4p which decorates the
spongiome (Cheng et al., 2023).
Following 5 hours of hairpin induction, Dop1p-mNeon still shows clear
localization to the CV bladder that is qualitatively similar to that in
non-induced or wildtype cells, and the bladder profiles are also similar
(Fig. 2A, compare left and right panels). However, by 8 hours of hairpin
induction the intensity of Dop1p bladder steady state labeling was
significantly decreased (Fig. 2B and S2). This change in the association
between Dop1p and the bladder membrane upon VPS8D knockdown could
also be seen at the level of dynamics. We previously used fluorescence
recovery after photobleaching (FRAP) to discover that Dop1p at the
bladder exchanges with a cytosolic pool (Cheng et al., 2023), as shown
in Figure 2C and 2D. That exchange was inhibited in VPS8Dknockdown cells, so that the rate of fluorescence recovery at the
bleached bladder was decreased (Fig. 2E, 2F and 2G). Lastly, the period
between contractions was lengthened in these VPS8D knockdown
cells (Fig. 2H). The mechanisms controlling bladder contraction are not
known, but one possibility is that bladder refilling is less efficient
in the mutant. Intriguingly, VPS8D hairpin induction had a clear
effect on the spongiome morphology. In particular, the volume occupied
by Vma4p-decorated tubules showed a marked decrease within 5 hours ofVPS8D -hairpin induction (Fig. 2I, 2J and S3), suggesting that
spongiome architecture depends more acutely than bladder structure on
the activity of Vps8Dp. Interestingly, these rapid changes in spongiome
structure upon VPS8D knockdown are similar to those seen after
cells are exposed to brief hypoosmotic challenge (Fig. S4).