Cellular location and temperature-dependent assembly of IncHI1 plasmid R27-encoded TrhC-associated conjugative transfer protein complexes

Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several minor states can be located quite precisely. The distribution of these minor states correlates with the electrostatic potential map around cytochrome P450cam. Whereas some minor states are on isolated positively charged patches, others are connected to the stereospecific site via positively charged paths. The existence of electrostatically favorable pathways between the stereospecific interaction site and the different minor states or lack thereof suggests a means to discriminate between productive and futile encounter states.

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CARTS biogenesis requires VAP–lipid transfer protein complexes functioning at the endoplasmic reticulum–Golgi interface

Molecular Biology of the Cell ◽

10.1091/mbc.e15-08-0599 ◽

2015 ◽

Vol 26 (25) ◽

pp. 4686-4699 ◽

Cited By ~ 30

Author(s):

Yuichi Wakana ◽

Richika Kotake ◽

Nanako Oyama ◽

Motohide Murate ◽

Toshihide Kobayashi ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Cell Surface ◽

Lipid Transfer Protein ◽

Protein Complexes ◽

Lipid Transfer ◽

Trans Golgi Network ◽

Transfer Protein ◽

Membrane Contact Sites ◽

Golgi Network

Vesicle-associated membrane protein–associated protein (VAP) is an endoplasmic reticulum (ER)-resident integral membrane protein that controls a nonvesicular mode of ceramide and cholesterol transfer from the ER to the Golgi complex by interacting with ceramide transfer protein and oxysterol-binding protein (OSBP), respectively. We report that VAP and its interacting proteins are required for the processing and secretion of pancreatic adenocarcinoma up-regulated factor, whose transport from the trans-Golgi network (TGN) to the cell surface is mediated by transport carriers called “carriers of the trans-Golgi network to the cell surface” (CARTS). In VAP-depleted cells, diacylglycerol level at the TGN was decreased and CARTS formation was impaired. We found that VAP forms a complex with not only OSBP but also Sac1 phosphoinositide phosphatase at specialized ER subdomains that are closely apposed to the trans-Golgi/TGN, most likely reflecting membrane contact sites. Immobilization of ER–Golgi contacts dramatically reduced CARTS production, indicating that association–dissociation dynamics of the two membranes are important. On the basis of these findings, we propose that the ER–Golgi contacts play a pivotal role in lipid metabolism to control the biogenesis of transport carriers from the TGN.

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Transcriptional network of the industrial hybrid Saccharomyces pastorianus reveals temperature-dependent allele expression bias and preferential orthologous protein assemblies

10.1101/2021.04.07.438844 ◽

2021 ◽

Author(s):

Soukaina Timouma ◽

Laura Natalia Balarezo-Cisneros ◽

Javier Pinto Aguirre ◽

Roberto De la Cerda ◽

Ursula M Bond ◽

...

Keyword(s):

Protein Complexes ◽

Temperature Response ◽

Protein Isoforms ◽

Temperature Dependent ◽

Transcriptional Signature ◽

Parental Allele ◽

Saccharomyces Pastorianus ◽

Transcriptional Pattern ◽

Cold Tolerant ◽

Hybrid Genes

Saccharomyces pastorianus is an industrial natural yeast evolved from different hybridisation events between the mesophilic S. cerevisiae and the cold-tolerant S. eubayanus. This complex aneuploid hybrid carries multiple copies of the parental alleles alongside specific hybrid genes and encodes for multiple protein isoforms which impart novel phenotypes, such as the strong ability to ferment at low temperature. These characteristics lead to agonistic or antagonistic competition for substrates and a plethora of biochemical activities, resulting in a unique cellular metabolism. Here, we investigated the transcriptional signature of the different orthologous alleles in S. pastorianus during temperature shifts. We identified temperature-dependent media-independent genes and showed that 35% have their regulation dependent on extracellular leucine uptake, suggesting an interplay between leucine metabolism and temperature response. The analysis of the expression of ortholog parental alleles unveiled that the majority of the genes express preferentially one parental allele over the other, and that S. eubayanus-like alleles are significantly over-represented among the genes involved in cold acclimatisation. The presence of functionally redundant parental alleles may impact on the nature of protein complexes established in the hybrid, where both parental alleles are competing. Our expression data indicate that the majority of the protein complexes established in the hybrid are likely to be either exclusively chimeric or uni-specific, and that the redundancy is discouraged, a scenario which fits well with the stoichiometric balance-hypothesis. This study offers a first overview of the transcriptional pattern of S. pastorianus and provide a rationalisation for its unique industrial traits at expression level.

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