scholarly journals Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane extension

PLoS Genetics ◽  
2021 ◽  
Vol 17 (8) ◽  
pp. e1009727
Author(s):  
Tsuyoshi S. Nakamura ◽  
Yasuyuki Suda ◽  
Kenji Muneshige ◽  
Yuji Fujieda ◽  
Yuuya Okumura ◽  
...  
Keyword(s):  
Double Mutant ◽  
De Novo ◽  
Adaptor Protein ◽  
Lipid Transfer ◽  
Contact Site ◽  
Contact Sites ◽  
Prospore Membrane ◽  
Protein Mutants ◽  
Membrane Extension ◽  
Multicopy Suppressors

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71–Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.

scholarly journals Dynamic localization of a yeast development–specific PP1 complex during prospore membrane formation is dependent on multiple localization signals and complex formation

2017 ◽  
Vol 28 (26) ◽  
pp. 3881-3895 ◽  
Author(s):  
Tsuyoshi S. Nakamura ◽  
Yumi Numajiri ◽  
Yuuya Okumura ◽  
Junji Hidaka ◽  
Takayuki Tanaka ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
De Novo ◽  
Developmental Process ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Membrane Structures ◽  
Prospore Membrane ◽  
Membrane Extension ◽  
And Function

During the developmental process of sporulation in Saccharomyces cerevisiae, membrane structures called prospore membranes are formed de novo, expand, extend, acquire a round shape, and finally become plasma membranes of the spores. GIP1 encodes a regulatory/targeting subunit of protein phosphatase type 1 that is required for sporulation. Gip1 recruits the catalytic subunit Glc7 to septin structures that form along the prospore membrane; however, the molecular basis of its localization and function is not fully understood. Here we show that Gip1 changes its localization dynamically and is required for prospore membrane extension. Gip1 first associates with the spindle pole body as the prospore membrane forms, moves onto the prospore membrane and then to the septins as the membrane extends, distributes around the prospore membrane after closure, and finally translocates into the nucleus in the maturing spore. Deletion and mutation analyses reveal distinct sequences in Gip1 that are required for different localizations and for association with Glc7. Binding to Glc7 is also required for proper localization. Strikingly, localization to the prospore membrane, but not association with septins, is important for Gip1 function. Further, our genetic analysis suggests that a Gip1–Glc7 phosphatase complex regulates prospore membrane extension in parallel to the previously reported Vps13, Spo71, Spo73 pathway.

scholarly journals Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation

Science ◽  
2020 ◽  
Vol 369 (6508) ◽  
pp. eaaz7714 ◽  
Author(s):  
Justyna Sawa-Makarska ◽  
Verena Baumann ◽  
Nicolas Coudevylle ◽  
Sören von Bülow ◽  
Veronika Nogellova ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
De Novo ◽  
Lipid Transfer ◽  
Related Protein ◽  
Membrane Formation ◽  
Selective Autophagy ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Phosphatidylinositol 3

Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)–containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast. We found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12–Atg5-Atg16 complex, which promoted Atg8 lipidation. Furthermore, we found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to the cargo via the Atg19-Atg11-Atg9 interactions. We thus propose that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum.

scholarly journals Competitive organelle-specific adaptors recruit Vps13 to membrane contact sites

2018 ◽  
Vol 217 (10) ◽  
pp. 3593-3607 ◽  
Author(s):  
Björn D.M. Bean ◽  
Samantha K. Dziurdzik ◽  
Kathleen L. Kolehmainen ◽  
Claire M.S. Fowler ◽  
Waldan K. Kwong ◽  
...  
Keyword(s):  
Repeat Region ◽  
Contact Site ◽  
Sorting Nexin ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Prospore Membrane ◽  
Membrane Contacts ◽  
Membrane Contact ◽  
Vacuolar Membranes ◽  
Mitochondrial Membrane Protein

The regulated expansion of membrane contact sites, which mediate the nonvesicular exchange of lipids between organelles, requires the recruitment of additional contact site proteins. Yeast Vps13 dynamically localizes to membrane contacts that connect the ER, mitochondria, endosomes, and vacuoles and is recruited to the prospore membrane in meiosis, but its targeting mechanism is unclear. In this study, we identify the sorting nexin Ypt35 as a novel adaptor that recruits Vps13 to endosomal and vacuolar membranes. We characterize an interaction motif in the Ypt35 N terminus and identify related motifs in the prospore membrane adaptor Spo71 and the mitochondrial membrane protein Mcp1. We find that Mcp1 is a mitochondrial adaptor for Vps13, and the Vps13–Mcp1 interaction, but not Ypt35, is required when ER-mitochondria contacts are lost. All three adaptors compete for binding to a conserved six-repeat region of Vps13 implicated in human disease. Our results support a competition-based model for regulating Vps13 localization at cellular membranes.

scholarly journals The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae

mSphere ◽  
2015 ◽  
Vol 1 (1) ◽  
Author(s):  
Yuuya Okumura ◽  
Tsuyoshi S. Nakamura ◽  
Takayuki Tanaka ◽  
Ichiro Inoue ◽  
Yasuyuki Suda ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Developmental Process ◽  
Spore Wall ◽  
Membrane Formation ◽  
Content Type ◽  
Prospore Membrane ◽  
Proper Size ◽  
Membrane Extension ◽  
Extension Analysis

ABSTRACT Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation, as an additional gene involved in prospore membrane extension. Genetic and cell biological analyses suggested that Spo73 functions on the prospore membrane with other factors in prospore membrane extension, counteracting the bending force of the prospore membrane. Spo73 is the first dysferlin domain-only protein ever analyzed. The dysferlin domain is conserved from yeast to mammals and is found in dysferlin proteins, which are involved in dysferlinopathy, although the precise function of the domain is unknown. Continued analysis of Spo73 will contribute to our understanding of the function of dysferlin domains and dysferlinopathy. Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation, as an additional gene involved in prospore membrane extension. Genetic and cell biological analyses suggested that Spo73 functions on the prospore membrane with other factors in prospore membrane extension, counteracting the bending force of the prospore membrane. Spo73 is the first dysferlin domain-only protein ever analyzed. The dysferlin domain is conserved from yeast to mammals and is found in dysferlin proteins, which are involved in dysferlinopathy, although the precise function of the domain is unknown. Continued analysis of Spo73 will contribute to our understanding of the function of dysferlin domains and dysferlinopathy.

scholarly journals SNAP iN, SNAP oUT—SNAREs at ER-PM Contact Sites

Contact ◽  
2020 ◽  
Vol 3 ◽  
pp. 251525642097958
Author(s):  
Neha Pratap Singh ◽  
Christian Vannier ◽  
Thierry Galli
Keyword(s):  
Protein Complexes ◽  
Short Review ◽  
Lipid Transfer ◽  
Contact Site ◽  
Lipid Transfer Proteins ◽  
Homologous Proteins ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Organelle Communication

Inter-organelle communication is essential for the exchange of cellular content in eukaryotes, particularly at membrane contact sites between the endoplasmic reticulum (ER) and the plasma membrane (PM). Accomplishing this critical task requires close positioning of the involved membranes via tether proteins and associated complexes. One such complex involves the SNAREs Sec22b and Syntaxin 1. Discovered to be interacting at the ER-PM membrane contact site (MCS), Sec22b-Stx1 forms a unique non-fusogenic bridge tethering the two membranes. Contrarily, SNAP25 was shown to be absent from the Sec22b-Stx1 complexes. Two recent studies focused on this interplay of SNARES and Lipid transfer proteins at MCSs. The Longin domain of Sec22b appeared to be the reason behind SNAP25’s exclusion from Sec22b-Stx1 assembly, and inclusion of E-Syts. It was also shown that yeast Sec9p and mammalian SNAP25 regulate ER-PM contact sites via their interaction with LTP OSBP-homologous proteins (ORP/OSH). In this following short review, we will take a closer look at the protein complexes involving SNAREs at MCSs and potential regulation by the Longin domain of Sec22b.

scholarly journals Serine palmitoyltransferase assembles at ER–mitochondria contact sites

2021 ◽  
Vol 5 (2) ◽  
pp. e202101278
Author(s):  
Mari J Aaltonen ◽  
Irina Alecu ◽  
Tim König ◽  
Steffany AL Bennett ◽  
Eric A Shoubridge
Keyword(s):  
De Novo ◽  
Outer Mitochondrial Membrane ◽  
Serine Palmitoyltransferase ◽  
Contact Site ◽  
Contact Sites ◽  
A Subunit ◽  
In Trans ◽  
Membrane Contact ◽  
And Function ◽  
Insight Into

The accumulation of sphingolipid species in the cell contributes to the development of obesity and neurological disease. However, the subcellular localization of sphingolipid-synthesizing enzymes is unclear, limiting the understanding of where and how these lipids accumulate inside the cell and why they are toxic. Here, we show that SPTLC2, a subunit of the serine palmitoyltransferase (SPT) complex, catalyzing the first step in de novo sphingolipid synthesis, localizes dually to the ER and the outer mitochondrial membrane. We demonstrate that mitochondrial SPTLC2 interacts and forms a complex in trans with the ER-localized SPT subunit SPTLC1. Loss of SPTLC2 prevents the synthesis of mitochondrial sphingolipids and protects from palmitate-induced mitochondrial toxicity, a process dependent on mitochondrial ceramides. Our results reveal the in trans assembly of an enzymatic complex at an organellar membrane contact site, providing novel insight into the localization of sphingolipid synthesis and the composition and function of ER–mitochondria contact sites.

Ady3p Links Spindle Pole Body Function to Spore Wall Synthesis in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1439-1450
Author(s):  
Mark E Nickas ◽  
Aaron M Neiman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Spindle Pole Body ◽  
Leading Edge ◽  
Spindle Pole ◽  
Spore Wall ◽  
Pole Body ◽  
Prospore Membrane ◽  
Sporulation Efficiency ◽  
Spore Walls

Abstract Spore formation in Saccharomyces cerevisiae requires the de novo synthesis of prospore membranes and spore walls. Ady3p has been identified as an interaction partner for Mpc70p/Spo21p, a meiosis-specific component of the outer plaque of the spindle pole body (SPB) that is required for prospore membrane formation, and for Don1p, which forms a ring-like structure at the leading edge of the prospore membrane during meiosis II. ADY3 expression has been shown to be induced in midsporulation. We report here that Ady3p interacts with additional components of the outer and central plaques of the SPB in the two-hybrid assay. Cells that lack ADY3 display a decrease in sporulation efficiency, and most ady3Δ/ady3Δ asci that do form contain fewer than four spores. The sporulation defect in ady3Δ/ady3Δ cells is due to a failure to synthesize spore wall polymers. Ady3p forms ring-like structures around meiosis II spindles that colocalize with those formed by Don1p, and Don1p rings are absent during meiosis II in ady3Δ/ady3Δ cells. In mpc70Δ/mpc70Δ cells, Ady3p remains associated with SPBs during meiosis II. Our results suggest that Ady3p mediates assembly of the Don1p-containing structure at the leading edge of the prospore membrane via interaction with components of the SPB and that this structure is involved in spore wall formation.

New horizons in mitochondrial contact site research

2020 ◽  
Vol 401 (6-7) ◽  
pp. 793-809
Author(s):  
Naama Zung ◽  
Maya Schuldiner
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Mechanisms ◽  
Contact Site ◽  
New Horizons ◽  
Contact Sites ◽  
Future Goals ◽  
Close Proximity ◽  
Recent Developments ◽  
Site Field ◽  
First Contact

AbstractContact sites, areas where two organelles are held in close proximity through the action of molecular tethers, enable non-vesicular communication between compartments. Mitochondria have been center stage in the contact site field since the discovery of the first contact between mitochondria and the endoplasmic reticulum (ER) over 60 years ago. However, only now, in the last decade, has there been a burst of discoveries regarding contact site biology in general and mitochondrial contacts specifically. The number and types of characterized contacts increased dramatically, new molecular mechanisms enabling contact formation were discovered, additional unexpected functions for contacts were shown, and their roles in cellular and organismal physiology were emphasized. Here, we focus on mitochondria as we highlight the most recent developments, future goals and unresolved questions in the field.

scholarly journals Seipin is required for converting nascent to mature lipid droplets

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Huajin Wang ◽  
Michel Becuwe ◽  
Benjamin E Housden ◽  
Chandramohan Chitraju ◽  
Ashley J Porras ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Lipid Synthesis ◽  
Human Cells ◽  
Lipid Transfer ◽  
Cellular Lipid ◽  
Contact Sites ◽  
Discrete Step

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation—the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.

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