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. 2017 Nov;16(11):1972-1989.
doi: 10.1074/mcp.RA117.000276. Epub 2017 Sep 8.

Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling

Zachary McBride  1 , Donglai Chen  2 , Christy Reick  3 , Jun Xie  2 , Daniel B Szymanski  4   5
Affiliations

Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling

Zachary McBride et al. Mol Cell Proteomics. 2017 Nov.

Abstract

Membrane-associated proteins are required for essential processes like transport, organelle biogenesis, and signaling. Many are expected to function as part of an oligomeric protein complex. However, membrane-associated proteins are challenging to work with, and large-scale data sets on the oligomerization state of this important class of proteins is missing. Here we combined cell fractionation of Arabidopsis leaves with nondenaturing detergent solubilization and LC/MS-based profiling of size exclusion chromatography fractions to measure the apparent masses of >1350 membrane-associated proteins. Our method identified proteins from all of the major organelles, with more than 50% of them predicted to be part of a stable complex. The plasma membrane was the most highly enriched in large protein complexes compared with other organelles. Hundreds of novel protein complexes were identified. Over 150 proteins had a complicated localization pattern, and were clearly partitioned between cytosolic and membrane-associated pools. A subset of these dual localized proteins had oligomerization states that differed based on localization. Our data set is an important resource for the community that includes new functionally relevant data for membrane-localized protein complexes that could not be predicted based on sequence alone. Our method enables the analysis of protein complex localization and dynamics, and is a first step in the development of a method in which LC/MS profile data can be used to predict the composition of membrane-associated protein complexes.

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Figures

Fig. 1.
Fig. 1.
Workflow for a proteomic analysis of membrane-associated protein complexes. Arabidopsis leaves were homogenized and a crude microsomal fraction was isolated. Microsomal proteins were solubilized in cholate and resolved by size exclusion chromatography. Fractions were collected, digested, and analyzed by LC-MS/MS. MaxQuant was used to identify the peptides and generate XIC abundance profiles. Peaks in the elution profiles were identified using Gaussian fitting. From the SEC profiles protein oligomerization predictions were made by calculating the ratio (Rapp) of the apparent mass (Mapp) determined by SEC to the monomer mass (Mmono) of the protein. Proteins with a Rapp ≥2 were predicted to form a putative complex. Contaminating soluble proteins were removed from the data set based their inability to sufficiently penetrate a sucrose gradient following ultracentrifugation (green box).
Fig. 2.
Fig. 2.
Cholate effectively solubilizes microsomal proteins. A, The soluble and membrane fractions were obtained by differential centrifugation, resolved on a SDS-PAGE gel by equal proportions and visualized by staining. S200 represents soluble proteins in the crude cytosol fraction. P200-elute 1 and p200-elute 2 were the supernatants from the respective washes. The microsomal fraction (P200) was resuspended in 4% cholate to solubilize membrane-associated proteins. After ultra-centrifugation the cholate-solubilized proteins were in the supernatant (P200-Cholate Sol.) and the insoluble proteins in the pellet (P200-cholate insol.). B, Western blot analysis of cholate solubilization of a panel of proteins with differing solubilities. Cytosolic PHOSPHOENOL-PYRUVATE CARBOXYLASE (PEPC), BINDING IMMUNOGLOBULIN PROTEIN (BIP), SEC21, and SPIKE1 (SPK1) (36) represent proteins that do not contain a transmembrane domain; SYP22 (52) has a single transmembrane domain, and H+ ATPase contains multiple transmembrane domains.
Fig. 3.
Fig. 3.
Reproducible apparent mass determinations for a previously uncharacterized set of microsomal proteins. A, A heat map of the Pearson correlation coefficients of the protein quantification signals in two biological replicates. The matrix was generated by Data Analysis and Extension Tool (DAnTE) (90). B, Protein coverage of proteins with a known and single localization based on the SUBAcon database: endomembrane system (Golgi, endoplasmic reticulum, plasma membrane, nucleus, peroxisome), plastid/mitochondria, or cytosolic/secreted. The number of proteins in this study are compared with two others that analyzed soluble proteins in Arabidopsis. C, The cholate solubilized fraction includes proteins with diverse functionalities predicted by Panther protein classes (91).
Fig. 4.
Fig. 4.
Gaussian fitting identifies multiple oligomerization states for individual proteins. A, The FERRITIN 4 profile with multiple peaks can be deconvolved into two individual peaks. B, Separation of an unresolved peak in the void from a resolved peak for the ATPase-related DNA REPAIR PROTEIN. C, The number of protein groups in which 1, 2, or 3 reproducible peaks were identified.
Fig. 5.
Fig. 5.
A high proportion of microsomal proteins are likely to exist as a stable complex. A, Hierarchical clustering analysis was performed based on the reproducible deconvoluted Gaussian fitted peaks for each protein normalized 0 to 1 in Bio1. Red represents the fractions of peak intensity and blue represents fractions were the protein was identified at a lesser intensity. B, Evidence for widespread oligomerization. For the proteins detected in this study, the distribution of the measured apparent masses (red) was compared with the calculated monomeric masses (blue). C, A scatter plot of the Rapp values for all of the proteins in the two biological replicates that had reproducible peaks. The gray area indicates the 949 proteins that are predicted to elute as a complex based on an Rapp threshold of 2 or greater.
Fig. 6.
Fig. 6.
Most conserved membrane-associated complexes are partially assembled. A, Arabidopsis orthologs to known metazoan protein complexes that are archived in the Corum database were identified and the deduced mass of the conserved fully assembled complex (red bars) was compared with the measured apparent masses (gray circles) of the subunits that correspond to the given metazoan complex. B, The elution profile of 4 subunits from the LSM complex. C, The elution profiles of the 3 subunits of the ESCRT-0 complex.
Fig. 7.
Fig. 7.
Identification of true membrane-associated protein complexes and their oligomerization state as a function of subcellular localization. A, Coomassie stained SDS-PAGE gels of the fractions collected from the microsomal (left) or S200 crude cytosolic fraction (right) that were layered and resolved on continuous 22–52% linear sucrose gradients. B, The raw XIC intensities for the protein abundance profiles from fractions 8–25 were subjected to hierarchical clustering. The intensity for each protein was normalized from 0–1 based on the max intensity in the replicate. Red lines show fractions of peak intensity with blue lines indicating fractions of lesser intensity. The predicted localization of the protein of interest is marked with a black dash based on GFP data (green columns) or SUBAcon consensus predictions (magenta). C, Oligomerization state as a function of known localization. The Rapp values of membrane-associated proteins were grouped by their SUBAcon subcellular localization. Degraded proteins were defined as those with an Rapp < 0.5; monomers 0.5≤ Rapp<2; small complexes 2≤ Rapp<10; and large complexes Rapp ≥10.
Fig. 8.
Fig. 8.
Many membrane-associated proteins have a dual localization with differing oligomerization states. A, The Mapp values for proteins that were defined as true membrane-associated and had been previously been analyzed in the soluble fraction independent of cholate solubilization (55). The red points indicate the proteins with a predicted shift in mass between the soluble and membrane fraction by having a ratio of the Mapp between the cytosol and membrane to be greater than 2 or less than 0.5. The blue points were not predicted to have a shift in mass. The black labeled arrow labels the NITRLASE1 data point. B, The abundance profiles of three chaperones coded as black (HIP1), green (CPN10), and red (HSBP) profiles were in larger complexes in the soluble fraction (solid lines) compared with their corresponding membrane-associated form (dashed lines). C, Three GRF/14-3-3 isoforms were identified as dual localized. The profiles in the membrane fraction reveal both a large complex with distinct masses and coeluting dimeric forms (dashed lines). The cytosolic fraction contained coeluting isoforms that migrated as trimers (solid lines). D, The elution profiles on the Superose column of the membrane-associated proteins that were identified by coIP-MS using the NITRILASE antibody. E, Elution profiles of published NITRILASE1 interactors in the soluble fraction from Aryal et al., 2017 (55).
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References

    1. Kush G. S. (2001) Green revolution: the way forward. Nat. Rev. Genet. 2, 815–822 - PubMed
    1. Hartwell L., Hopfield J., Leibler S., and Murray A. (1999) From molecular to modular cell biology. Nature 402, C47–C52 - PubMed
    1. Alberts B. (1998) The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92, 291–294 - PubMed
    1. Berg J. M., Tymoczko J. L., Gatto G. J. Jr, and Stryer L. (2015) Biochemistry (W. H. Freeman, New York: ). 8th Ed.
    1. Buchanan B. B., Gruissem W., and Jones R. L. (2015) Biochemistry and Molecular Biology of Plants (Whiley) 2nd Ed.

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