Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome

Abstract

Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease gene.

A role for Mitochondrial Rho GTPase 1 (MIRO1) in motility and membrane dynamics of peroxisomes

“…A dual mitochondrial and peroxisomal localisation of MIRO1 was confirmed by immunofluorescence after expression of Myc‐MIRO1 in COS‐7 cells (Figure 1A). Furthermore, we previously reported endogenous MIRO1 in highly purified peroxisomal and mitochondrial fractions,34 in agreement with proteomics data 35, 36…”

“…Furthermore, we previously reported endogenous MIRO1 in highly purified peroxisomal and mitochondrial fractions, 34 in agreement with proteomics data. 35,36 The targeting of all known TA proteins to peroxisomes requires the peroxisomal import receptor/chaperone PEX19. 34 For MIRO1, PEX19 binding was shown by immunoprecipitation after coexpression of Myc-MIRO1 and HA-PEX19 in COS-7 cells ( Figure 1B) suggesting a role for PEX19 in the targeting of MIRO1 to peroxisomes.…”

Using whole‐exome sequencing to investigate the genetic bases of lysosomal storage diseases of unknown etiology

“…In addition, TBCK was implicated in the mTOR pathway (Liu et al, 2013), which regulates lysosomal biogenesis and function (Kinghorn et al, 2017). Therefore, although subcellular localization analysis (Jadot et al, 2017) indicates that TBCK does not reside within the lysosome, these studies suggest that TBCK may control some aspects of lysosomal function and could result in phenotypes in CABMHF3.…”

“…Val181Leu mutation. SLC31A1 was targeted for analysis based on its dual localization to both the lysosome and to the plasma membrane, and we have reported previously on the variants in this patient (Jadot et al, 2017). While p.Arg90Ter is a clear null, further functional analysis to test the cellular copper uptake are necessary to demonstrate the effect of these two variants together.…”

SNAT7 is the primary lysosomal glutamine exporter required for extracellular protein-dependent growth of cancer cells

“…It should be stressed that although luminal acidity stimulated lysosomal [ 3 H]glutamine uptake under the artificial conditions of our countertransport assay, our in situ TFEB-based assay clearly shows that SNAT7 operates in the export direction in live cells. A very recent proteomic study of multiple subcellular compartments from rat liver also identified lysosomes as the major, if not exclusive, cellular location of SNAT7 (38).…”