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DDX6 is an abundant DEAD-box helicase associated with various complexes involved in mRNA decay and repression. Its interactome in human cells was analyzed to identify its most prominent partners. Among them, three proteins were essential for P-body assembly in all tested conditions: DDX6, 4E-T, and LSM14A., P-bodies are cytoplasmic ribonucleoprotein granules involved in posttranscriptional regulation. DDX6 is a key component of their assembly in human cells. This DEAD-box RNA helicase is known to be associated with various complexes, including the decapping complex, the CPEB repression complex, RISC, and the CCR4/NOT complex. To understand which DDX6 complexes are required for P-body assembly, we analyzed the DDX6 interactome using the tandem-affinity purification methodology coupled to mass spectrometry. Three complexes were prominent: the decapping complex, a CPEB-like complex, and an Ataxin2/Ataxin2L complex. The exon junction complex was also found, suggesting DDX6 binding to newly exported mRNAs. Finally, some DDX6 was associated with polysomes, as previously reported in yeast. Despite its high enrichment in P-bodies, most DDX6 is localized out of P-bodies. Of the three complexes, only the decapping and CPEB-like complexes were recruited into P-bodies. Investigation of P-body assembly in various conditions allowed us to distinguish required proteins from those that are dispensable or participate only in specific conditions. Three proteins were required in all tested conditions: DDX6, 4E-T, and LSM14A. These results reveal the variety of pathways of P-body assembly, which all nevertheless share three key factors connecting P-body assembly to repression.

LOA1, a yeast member of the glycerolipid acyltransferase family, encodes a novel lysophosphatidic acid acyltransferase associated with lipid droplets (LDs) and involved in triacylglycerol (TAG) accumulation. Loa1p, recruited during LD formation, preferentially directs oleic acid–containing phosphatidic acid species into the TAG biosynthetic pathway., For many years, lipid droplets (LDs) were considered to be an inert store of lipids. However, recent data showed that LDs are dynamic organelles playing an important role in storage and mobilization of neutral lipids. In this paper, we report the characterization of LOA1 (alias VPS66, alias YPR139c), a yeast member of the glycerolipid acyltransferase family. LOA1 mutants show abnormalities in LD morphology. As previously reported, cells lacking LOA1 contain more LDs. Conversely, we showed that overexpression results in fewer LDs. We then compared the lipidome of loa1Δ mutant and wild-type strains. Steady-state metabolic labeling of loa1Δ revealed a significant reduction in triacylglycerol content, while phospholipid (PL) composition remained unchanged. Interestingly, lipidomic analysis indicates that both PLs and glycerolipids are qualitatively affected by the mutation, suggesting that Loa1p is a lysophosphatidic acid acyltransferase (LPA AT) with a preference for oleoyl-CoA. This hypothesis was tested by in vitro assays using both membranes of Escherichia coli cells expressing LOA1 and purified proteins as enzyme sources. Our results from purification of subcellular compartments and proteomic studies show that Loa1p is associated with LD and active in this compartment. Loa1p is therefore a novel LPA AT and plays a role in LD formation.

HSF2 regulates proteostasis capacity against febrile-range thermal stress, which provides temperature-dependent mechanisms of cellular adaptation to thermal stress. Furthermore, HSF2 has a strong impact on disease progression of Huntington's disease R6/2 mice, suggesting that it could be a promising therapeutic target for protein misfolding diseases., Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.