Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution.

Actively maintained close appositions between organelle membranes, also known as contact sites, enable the efficient transfer of biomolecules between cellular compartments. Several such sites have been described as well as their tethering machineries. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize contact site proteomes, we established a high-throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, several of which are poorly characterized, on the background of 1165 strains expressing a mCherry-tagged yeast protein that has a cellular punctate distribution (a hallmark of contact sites), under regulation of the strong TEF2 promoter. By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified three homologs of Vps13 and Atg2 that are residents of multiple contact sites. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell. Overall, our analysis expands the universe of contact site residents and effectors and creates a rich database to mine for new functions, tethers, and regulators.

Pls1 Is a Peroxisomal Matrix Protein with a Role in Regulating Lysine Biosynthesis.

Peroxisomes host essential metabolic enzymes and are crucial for human health and survival. Although peroxisomes were first described over 60 years ago, their entire proteome has not yet been identified. As a basis for understanding the variety of peroxisomal functions, we used a high-throughput screen to discover peroxisomal proteins in yeast. To visualize low abundance proteins, we utilized a collection of strains containing a peroxisomal marker in which each protein is expressed from the constitutive and strong TEF2 promoter. Using this approach, we uncovered 18 proteins that were not observed in peroxisomes before and could show their metabolic and targeting factor dependence for peroxisomal localization. We focus on one newly identified and uncharacterized matrix protein, Ynl097c-b, and show that it localizes to peroxisomes upon lysine deprivation and that its localization to peroxisomes depends on the lysine biosynthesis enzyme, Lys1. We demonstrate that Ynl097c-b affects the abundance of Lys1 and the lysine biosynthesis pathway. We have therefore renamed this protein Pls1 for Peroxisomal Lys1 Stabilizing 1. Our work uncovers an additional layer of regulation on the central lysine biosynthesis pathway. More generally it highlights how the discovery of peroxisomal proteins can expand our understanding of cellular metabolism.

The Fast and the Furious: Golgi Contact Sites.

Contact sites are areas of close apposition between two membranes that coordinate nonvesicular communication between organelles. Such interactions serve a wide range of cellular functions from regulating metabolic pathways to executing stress responses and coordinating organelle inheritance. The past decade has seen a dramatic increase in information on certain contact sites, mostly those involving the endoplasmic reticulum. However, despite its central role in the secretory pathway, the Golgi apparatus and its contact sites remain largely unexplored. In this review, we discuss the current knowledge of Golgi contact sites and share our thoughts as to why Golgi contact sites are understudied. We also highlight what exciting future directions may exist in this emerging field.

Sarcopenia defined by muscle quality rather than quantity predicts complications following laparoscopic right hemicolectomy.

PURPOSE: While sarcopenia has prognostic value in elective colorectal surgery for predicting peri-operative morbidity and mortality, its role in elective laparoscopic surgery is poorly defined. METHODS: A retrospective single-center analysis of patients undergoing elective laparoscopic right hemicolectomy for adenocarcinoma between January 2010 and December 2016. Univariate analysis compared the robustness of total psoas index (TPI) with Hounsfield unit average calculation (HUAC) calculated from pre-operative CT imaging in predicting post-operative complications. Multivariate analysis compared these measures with American Society of Anesthesiologists (ASA) grade and Charlson scores in predicting post-operative complications. RESULTS: Of the 580 patients identified, 185 met the inclusion criteria (91 males and 94 females, with a median age of 68). Using TPI and HUAC, 46 and 44 patients respectively were identified as sarcopenic, including 18 patients that were identified by both measures. HUAC-defined sarcopenia was significantly associated with pre-operative comorbidities, peri-operative mortality, and a greater incidence of respiratory, cardiac, and serious post-operative complications (Clavien-Dindo ≥ 3). Those with HUAC-defined sarcopenia aged > 75 were at particular risk of morbidity (OR 5.52, p = 0.002). No such relationships were found with TPI-defined sarcopenia. Only HUAC remained predictive of post-operative complications on multivariate analysis. CONCLUSION: Sarcopenia is a novel methodology for stratifying surgical risk in elective colorectal cancer surgery. HUAC has a high prognostic accuracy for the prediction of complications following laparoscopic colorectal surgery compared with TPI, ASA grade, and Charlson score.