How and why intralumenal membrane fragments form during vacuolar lysosome fusion

Abstract

When vacuolar lysosomes fuse, an intralumenal membrane fragment is produced and degraded by hydrolyses. How or why this fragment forms is not entirely understood. We show that the fusion machinery regulates stalk expansion during lipid bilayer fusion to create or eliminate fragments, affecting lysosome morphology and transporter protein turnover.

Distinct features of multivesicular body‐lysosome fusion revealed by a new cell‐free content‐mixing assay

“…We then imaged these organelle preparations using transmission electron microscopy (TEM) and observed structures resembling MVBs, some of which were in close contact with vacuole membranes, a prerequisite for fusion ( Figure 1D). 36 We also observed the presence of our fusion probes (Pep12-Fos-Gs-ω or CPY50-Jun-Gs-α) in this fraction by western blot analysis ( Figure 1E). Markers of both MVBs (Vps10, endogenous Pep12) and vacuoles (Nyv1, endogenous CPY) and fusogenic proteins (eg, Ypt7, Vps41, Vps33 and Nyv1) were also present confirming that both organelles are found in this preparation and are likely fusogenic.…”

“…Pep12‐GFP is present in the preparation and localizes to small puncta adjacent to vacuole membranes reminiscent of MVB structures, whereas CPY50‐GFP is found within the lumen of isolated vacuoles, consistent with their distributions in living cells. We then imaged these organelle preparations using transmission electron microscopy (TEM) and observed structures resembling MVBs, some of which were in close contact with vacuole membranes, a prerequisite for fusion (Figure D) . We also observed the presence of our fusion probes (Pep12‐Fos‐Gs‐ω or CPY50‐Jun‐Gs‐α) in this fraction by western blot analysis (Figure E).…”

“…Isolated organelles were processed for TEM as described previously . In brief, fusion reactions were incubated at 27°C for 30 minutes, organelles were gently pelleted (5000 g for 5 minutes) at 4°C and immediately fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight at 4°C.…”

SNARE ‐mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle

“…In vitro, many proteins and lipids involved in vacuole fusion concentrate in a vertex ring surrounding the contact zone between vacuoles (Wang et al, 2002;Fratti et al, 2004;Karunakaran et al, 2012;Mattie et al, 2017;McNally et al, 2017). In order to explore in vivo whether vacuoles are connected through a single fusion zone, or by several dispersed ones, we followed the spatial distribution of FM4-64 FRAP.…”

“…Tethering, governed by coordinated action of the Rab‐GTPase Ypt7 and the HOPS complex, permits the formation of trans‐SNARE complexes (Mayer & Wickner, ; Price et al , ; Zick & Wickner, ; Orr et al , ; Lürick et al , ). These, together with V 0 proteolipids, induce lipid mixing (Peters et al , ; Reese et al , ; Strasser et al , ; Desfougères et al , ; Mattie et al , ). Content mixing requires several trans‐SNARE complexes (D'Agostino et al , ) and their anchoring in the membrane through peptidic transmembrane domains in helical continuity with the SNARE domain (Pieren et al , ).…”