scholarly journals Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane

2004 ◽  
Vol 167 (6) ◽  
pp. 1099-1112 ◽  
Author(s):  
Marcello Marelli ◽  
Jennifer J. Smith ◽  
Sunhee Jung ◽  
Eugene Yi ◽  
Alexey I. Nesvizhskii ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Large Scale ◽  
Secretory Pathway ◽  
Subcellular Fractionation ◽  
Membrane Dynamics ◽  
Small Gtpase ◽  
Quantitative Mass Spectrometry ◽  
Actin Organization ◽  
Associated Proteins ◽  
Peroxisome Membrane

We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

scholarly journals Distinct Roles for the Yeast Phosphatidylinositol 4-Kinases, Stt4p and Pik1p, in Secretion, Cell Growth, and Organelle Membrane Dynamics

2000 ◽  
Vol 11 (8) ◽  
pp. 2673-2689 ◽  
Author(s):  
Anjon Audhya ◽  
Michelangelo Foti ◽  
Scott D. Emr
Keyword(s):  
Cell Growth ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Membrane Dynamics ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Restrictive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cytoskeleton Organization ◽  
Yeast Viability

The yeast Saccharomyces cerevisiae possesses two genes that encode phosphatidylinositol (PtdIns) 4-kinases,STT4 and PIK1. Both gene products phosphorylate PtdIns at the D-4 position of the inositol ring to generate PtdIns(4)P, which plays an essential role in yeast viability because deletion of either STT4 orPIK1 is lethal. Furthermore, although both enzymes have the same biochemical activity, increased expression of either kinase cannot compensate for the loss of the other, suggesting that these kinases regulate distinct intracellular functions, each of which is required for yeast cell growth. By the construction of temperature-conditional single and double mutants, we have found that Stt4p activity is required for the maintenance of vacuole morphology, cell wall integrity, and actin cytoskeleton organization. In contrast, Pik1p is essential for normal secretion, Golgi and vacuole membrane dynamics, and endocytosis. Strikingly,pik1tscells exhibit a rapid defect in secretion of Golgi-modified secretory pathway cargos, Hsp150p and invertase, whereas stt4tscells exhibit no detectable secretory defects. Both single mutants reduce PtdIns(4)P by ∼50%; however,stt4ts/pik1tsdouble mutant cells produce more than 10-fold less PtdIns(4)P as well as PtdIns(4,5)P2. The aberrant Golgi morphology found in pik1tsmutants is strikingly similar to that found in cells lacking the function of Arf1p, a small GTPase that is known to regulate multiple membrane trafficking events throughout the cell. Consistent with this observation, arf1 mutants exhibit reduced PtdIns(4)P levels. In contrast, diminished levels of PtdIns(4)P observed in stt4tscells at restrictive temperature result in a dramatic change in vacuole size compared with pik1tscells and persistent actin delocalization. Based on these results, we propose that Stt4p and Pik1p act as the major, if not the only, PtdIns 4-kinases in yeast and produce distinct pools of PtdIns(4)P and PtdIns(4,5)P2that act on different intracellular membranes to recruit or activate as yet uncharacterized effector proteins.

scholarly journals A review on subcellular or organellar proteomics with special reference to apicomplexan parasites

1970 ◽  
pp. 1-7
Author(s):  
AMAM Zonaed Siddiki ◽  
MB Hossain ◽  
ASM Lutful Ahasan
Keyword(s):  
Mass Spectrometry ◽  
Special Reference ◽  
Key Words ◽  
Large Scale ◽  
Subcellular Fractionation ◽  
Apicomplexan Parasites ◽  
Proteomic Investigation ◽  
Analytical Tools ◽  
Subcellular Proteomics

Despite several well-known limitations, mass spectrometry-based proteomics is still performing important role for post-genomic investigations. As large-scale proteomic investigation of whole organism or cell has been found more complex with available analytical tools, subcellular fractionation prior to mass spectrometry is becoming more useful approach now-a-days. In this review, an attempt has been made to summarize all such subcellular or organellar proteomic investigations performed to date with its implications for apicomplexan parasites. Key words: Subcellular proteomics, parasite, mass spectrometry, fractionation DOI = 10.3329/bjvm.v5i1.1301 Bangl. J. Vet. Med. (2007). 5 (1 & 2): 01- 07

scholarly journals Role of clathrin in dense core vesicle biogenesis

2017 ◽  
Vol 28 (20) ◽  
pp. 2676-2685 ◽  
Author(s):  
Bhavani S. Sahu ◽  
Paul T. Manna ◽  
James R. Edgar ◽  
Robin Antrobus ◽  
Sushil K. Mahata ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Subcellular Fractionation ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Bioactive Molecules ◽  
Dense Core Vesicle ◽  
Data Sets ◽  
Protein Loss ◽  
Associated Proteins

The dense core vesicles (DCVs) of neuroendocrine cells are a rich source of bioactive molecules such as peptides, hormones, and neurotransmitters, but relatively little is known about how they are formed. Using fractionation profiling, a method that combines subcellular fractionation with mass spectrometry, we identified ∼1200 proteins in PC12 cell vesicle-enriched fractions, with DCV-associated proteins showing distinct profiles from proteins associated with other types of vesicles. To investigate the role of clathrin in DCV biogenesis, we stably transduced PC12 cells with an inducible short hairpin RNA targeting clathrin heavy chain, resulting in ∼85% protein loss. DCVs could still be observed in the cells by electron microscopy, but mature profiles were approximately fourfold less abundant than in mock-treated cells. By quantitative mass spectrometry, DCV-associated proteins were found to be reduced approximately twofold in clathrin-depleted cells as a whole and approximately fivefold in vesicle-enriched fractions. Our combined data sets enabled us to identify new candidate DCV components. Secretion assays revealed that clathrin depletion causes a near-complete block in secretagogue-induced exocytosis. Taken together, our data indicate that clathrin has a function in DCV biogenesis beyond its established role in removing unwanted proteins from the immature vesicle.

scholarly journals The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin

2021 ◽  
Author(s):  
Charlotte Repton ◽  
C Fiona Cullen ◽  
Mariana FA Costa ◽  
Christos Spanos ◽  
Juri Rappsilber ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Regulatory Mechanism ◽  
Mass Spectrometry Data ◽  
Quantitative Mass Spectrometry ◽  
Microtubule Associated Proteins ◽  
Chromosomal Passenger ◽  
Associated Proteins ◽  
And Function ◽  
Systematic Identification ◽  
Docking Protein

Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. We previously uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. Here we report systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, we identified proteins or complexes whose ability to binding microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. We showed that 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function. The mass spectrometry data are available from ProteomeXchange, identifier ID to be provided when available, PXD000xxx.

scholarly journals Stable isotope dimethyl labelling for quantitative proteomics and beyond

2016 ◽  
Vol 374 (2079) ◽  
pp. 20150364 ◽  
Author(s):  
Jue-Liang Hsu ◽  
Shu-Hui Chen
Keyword(s):  
Mass Spectrometry ◽  
Stable Isotope ◽  
Quantitative Proteomics ◽  
Large Scale ◽  
Protein Identification ◽  
Cost Effective ◽  
Quantitative Mass Spectrometry ◽  
Post Translational Modifications ◽  
Labelling Method ◽  
Identification And Characterization

Stable-isotope reductive dimethylation, a cost-effective, simple, robust, reliable and easy-to- multiplex labelling method, is widely applied to quantitative proteomics using liquid chromatography-mass spectrometry. This review focuses on biological applications of stable-isotope dimethyl labelling for a large-scale comparative analysis of protein expression and post-translational modifications based on its unique properties of the labelling chemistry. Some other applications of the labelling method for sample preparation and mass spectrometry-based protein identification and characterization are also summarized. This article is part of the themed issue ‘Quantitative mass spectrometry’.

Algorithm of combining chromatography mass spectrometry-untargeted profiling and multivariate analysis for identification of marker-substances in samples of complex composition

2020 ◽  
Vol 86 (7) ◽  
pp. 12-19
Author(s):  
I. V. Plyushchenko ◽  
D. G. Shakhmatov ◽  
I. A. Rodin
Keyword(s):  
Mass Spectrometry ◽  
Multivariate Analysis ◽  
Large Scale ◽  
Complex Composition ◽  
Unified Protocol ◽  
Chromatography Mass Spectrometry ◽  
Marker Substances ◽  
Selection Testing ◽  
Untargeted Profiling

A viral development of statistical data processing, computing capabilities, chromatography-mass spectrometry, and omics technologies (technologies based on the achievements of genomics, transcriptomics, proteomics, metabolomics) in recent decades has not led to formation of a unified protocol for untargeted profiling. Systematic errors reduce the reproducibility and reliability of the obtained results, and at the same time hinder consolidation and analysis of data gained in large-scale multi-day experiments. We propose an algorithm for conducting omics profiling to identify potential markers in the samples of complex composition and present the case study of urine samples obtained from different clinical groups of patients. Profiling was carried out by the method of liquid chromatography mass spectrometry. The markers were selected using methods of multivariate analysis including machine learning and feature selection. Testing of the approach was performed using an independent dataset by clustering and projection on principal components.

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