The yeast Batten disease orthologue Btn1 controls endosome–Golgi retrograde transport via SNARE assembly

Btn1 controls endosome–Golgi retrograde sorting by regulating SNARE phosphorylation and assembly

Heterologous aggregates promote de novo prion appearance via more than one mechanism.

Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [PSI+], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN+], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN+] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI+][PIN+] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI+], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN+], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI+]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI+] induction in the absence of [PIN+]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI+], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI+] aggregates in a [PIN+]-independent pathway

Overexpressed Mod5 does not interact with Sup35 during [<i>PSI⁺</i>] induction in the absence of Rnq1.

<p><b>A.</b> Mod5 did not form visible aggregates although Sup35 newly appearing aggregates formed. Mod5-GFP (p2061) and Sup35NM-RFP (p2018) were simultaneously overexpressed in 74D-694 <i>rnq1Δ</i> cells with 2% Gal for the indicated times. Mod5-GFP remained diffuse although Sup35NM–RFP formed dots and rings (data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004814#pgen-1004814-t004" target="_blank">Table 4</a>). <b>B.</b> Mod5 did not form a close physical interaction with Sup35 during [<i>PSI⁺</i>] induction in the absence of [<i>PIN⁺</i>]. Mod5-VN (p2170) and Sup35NM-VC (p1892) were simultaneously overexpressed in 74D-694 [<i>pin^-</i>] (top) and [<i>PIN⁺</i>] (bottom) cells with 2% Gal for the indicated times. No fluorescence was detected in [<i>pin^-</i>], but Sup35-Mod5 dots appeared in [<i>PIN⁺</i>] cells mostly near the vacuole (arrows) following the diffuse fluorescence. (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004814#pgen.1004814.s011" target="_blank">S11B Fig.</a>)</p

Data for the aggregation of Sup35NM-RFP in <i>HSP42-GFP</i> cells shown in Fig. 3.

<p>BY4741 [<i>PIN⁺</i>][<i>psi^-</i>] <i>HSP42-GFP</i> cells were grown in 2% Gal to overexpress Sup35NM-RFP (from p2017). Data for the florescence of only Sup35NM-RFP is tabulated. Hsp42-GFP was always seen as 1 big spot near the vacuole in all cells.</p><p>Data for the aggregation of Sup35NM-RFP in <i>HSP42-GFP</i> cells shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004814#pgen-1004814-g003" target="_blank">Fig. 3</a>.</p

Hsp104 is required for the <i>de novo</i> [<i>PSI⁺</i>] induction.

<p><b>A.</b> Absence of Hsp104 dramatically reduces the frequency of <i>de novo</i> Sup35 aggregates. Sup35NM with a short tail of hydrophobic residues (Sup35NM-mt) was overexpressed from p1984 by growing <i>hsp104Δ</i> (<i>GF844</i>) or [<i>pin^-</i>] <i>HSP104</i> (<i>GF658</i>) cells with endogenous <i>SUP35-GFP</i> in 2% Gal. Sup35-GFP remained mostly diffuse in <i>hsp104Δ</i> cells with a few cells with short lines located at the cell membrane, but formed dots and short lines throughout the cytoplasm in [<i>pin^-</i>] <i>HSP104</i> cells after 48 h of induction. <b>B.</b> Hsp104 is necessary to induce Sup35 newly appearing rings and lines during Pin4C-promoted [<i>PSI⁺</i>] induction. Sup35NM-GFP and Pin4C-RFP were respectively co-overexpressed from p1181 and p1708, by growing <i>hsp104Δ</i> (L1802, or L1803), <i>HSP104</i> [<i>pin^-</i>] (L2910) or <i>HSP104</i> [<i>PIN⁺</i>] (L1749) cells in 50 µM CuSO₄ and 2% Gal. In the absence of Hsp104, Sup35NM-GFP failed to form any aggregates in contrast to <i>HSP104</i> cells. <b>C.</b> Inhibition of Hsp104 does not induce [<i>PSI⁺</i>] <i>de novo</i>. Sup35NM-GFP and Pin4C-RFP were co-overexpressed respectively from p1181 and p1708, by growing <i>HSP104</i> [<i>pin^-</i>] (L2910) cells in 50 µM CuSO₄, 2% Gal and with or without 10 mM GuHCl. In comparison to dots and ring formation by Sup35NM-GFP in the absence of GuHCl (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004814#pgen-1004814-g008" target="_blank">Fig. 8B</a>), Sup35NM-GFP never formed rings/lines in the presence of GuHCl. 2.8% of cells had dots and 97.2% of cells were diffuse. None of cells grown in the presence of GuHCl induced [<i>PSI⁺</i>]. The pictures were taken 72 h after the addition of copper, galactose and GuHCl to the growth media.</p

Models we propose to explain <i>de novo</i> Sup35 aggregation.

<p><b>A.</b> The stages of the induced appearance of [<i>PSI⁺</i>] <i>de novo</i>. Induction of [<i>PSI⁺</i>] by overexpression of fluorescently-labeled Sup35 in [<i>PIN⁺</i>][<i>psi^-</i>] cells displays early Sup35 fluorescent foci ranging from one to a few dots, one of which is located near the vacuole (shown as darker green). At this perivacuolar Hsp42 protein deposit site, preexisting heterologous aggregates such as [<i>PIN⁺</i>] or Pin4C cross-seed the <i>de novo</i> aggregation of Sup35 (see panel B). Later, short lines appear to emanate from this dot towards the periphery of the cell while dots away from the vacuole disappear. Peripheral rings appear next, followed by perivacuolar internal rings. <b>B.</b> The cross-seeding model to explain how heterologous aggregates facilitate <i>de novo</i> Sup35 aggregation. Preexisting [<i>PIN⁺</i>]/Pin4C aggregates (shown in red ‘L’ shape) located at the Hsp42 protein deposit near the vacuole physically interact with soluble misfolded Sup35 proteins (shown as purple marbles). This interaction causes Sup35 to form oligomers. Then, Sup35 grows in length in a homotypic manner. Based on our data, we propose that [<i>PIN⁺</i>]/Pin4C aggregates can also grow in length laterally guided by Sup35 fibrils. Over time, laterally growing [<i>PIN⁺</i>]/Pin4C aggregates are less frequent. For simplicity, only two fibrils are depicted.</p

Excess Pin4C temporarily colocalize with Sup35 aggregates during [<i>PSI⁺</i>] induction in the absence of Rnq1.

<p>Pin4C-RFP (p1708) and Sup35NM-GFP (p1951) were simultaneously overexpressed in 74D-694 <i>rnq1Δ</i> cells with 2% Gal for the indicated times. While Sup35NM-GFP early dots and rings (up to 48 h) colocalized with Pin4C-RFP, Pin4C-RFP lost colocalization with Sup35 rings later (72 h) (data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004814#pgen-1004814-t003" target="_blank">Table 3</a>).</p

Quantitative data for the aggregation of fluorescently-tagged Sup35 shown in Fig. 1.

<p>74D-694 [<i>PIN⁺</i>][<i>psi^-</i>] cells were grown in 2% Gal to overexpress Sup35NM-GFP (from p1951) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004814#pgen-1004814-g001" target="_blank">Fig. 1A</a>); or in 0.2% Gal to co-overexpress Sup35NM-VN (from p1893) and Sup35NM-VC (from p1892) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004814#pgen-1004814-g001" target="_blank">Fig. 1C</a>). 74D-694 [<i>PIN⁺</i>][<i>psi^-</i>] <i>SUP35-GFP</i> cells were grown in 2% Gal to overexpress Sup35NM (from p2036) (in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004814#pgen-1004814-g001" target="_blank">Figure 1B</a>) or Sup35 (from p743) (in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004814#pgen.1004814.s001" target="_blank">S1A Fig.</a>).</p><p>Quantitative data for the aggregation of fluorescently-tagged Sup35 shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004814#pgen-1004814-g001" target="_blank">Fig. 1</a>.</p

Sup35 aggregates initially appear near the vacuole, from which short lines extend to the periphery to form rings.

<p>Sup35NM-RFP was overexpressed from p2017 by growing [<i>PIN⁺</i>] BY4741 cells with endogenous <i>HSP42-GFP</i> in 2% Gal. Time shown is after the addition of 2% Gal (see details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004814#pgen-1004814-t002" target="_blank">Table 2</a>).</p

Yeast strains used in this study.

a<p><i>YFG</i>: Your Favorite Gene.</p><p>Yeast strains used in this study.</p