Genome-wide Analysis Relating Expression Level with Protein Subcellular Localization

Amar Drawid1, *, Ronald Jansen1, * & Mark Gerstein1, 2, †

Department of Molecular Biophysics & Biochemistry1 and Computer Science2
266 Whitney Avenue, Yale University
PO Box 208114, New Haven, CT 06520
(203) 432-6105, FAX (360) 838-7861

Amar.Drawid@yale.edu
Ronald.Jansen@yale.edu
Mark.Gerstein@yale.edu

* These authors contributed equally to this work.
† Corresponding author

Revised version (000803) sent to Trends in Genetics

Abstract

We investigate the relationship between protein subcellular localization and gene expression for a variety of whole-genome expression data sets.  We find high expression levels for cytoplasmic proteins and low ones for nuclear and membrane proteins. Excreted proteins have large fluctuations in expression level over various time courses.  Our results can be interpreted in terms of protein structure and function.  Detailed statistics are at http://bioinfo.mbb.yale.edu/genome/expression.

Main Text

The recent advent of experiments that measure gene expression levels (mRNA transcript abundance levels) on a genome-wide scale allows a comprehensive view of gene activity patterns in cells.  For instance, these experiments have demonstrated that the expression patterns of many functionally related genes are similar1-8.  Here we show for yeast that expression levels in these experiments are clearly correlated with the subcellular localization of the corresponding protein.  Furthermore, this correlation can be interpreted in terms of broad classes of protein structures and functions.

We scaled the expression levels generated by a range of techniques (gene chip, SAGE, cDNA microarray) for yeast in a variety of conditions into a common framework and cross-referenced them with the known localizations of ~2000 yeast proteins found in the MIPS9 and YPD10 databases. (Further details are given in the caption to figure 1 and on the associated website, http://bioinfo.mbb.yale.edu/genome/expression.) 

ABSOLUTE EXPRESSION LEVELS. As shown in figure 1, high expression levels can be observed for cytoplasmic proteins, low levels for nuclear and membrane proteins, and middling levels for secretory pathway proteins  -- i.e., those secreted or in the endoplasmic reticulum (ER) and golgi.

Figure 2 shows a more detailed representation of the absolute expression levels in figure 1. We chose the data set of Holstege et al. as a reference because it results from the careful averaging over many experiments. Figure 2a shows simplified box-plot representations of the underlying distributions of expression levels for each of the different subcellular compartments for this data set.  As is evident, for each compartment there is an appreciable spread of expression levels and the distributions for the compartments with the highest expression levels are more spread out.  

Full representations of each expression level distribution are shown on our website. These show that for all compartments, the distributions are roughly exponential, although the tails are somewhat longer.  The exponential shape may reflect the fact that many genes are expressed at a basal level whereas a smaller number are highly active in the particular state of the cell.

Figure 2b shows key statistics based on the box plots in figure 2a for the Holstege et al. data set. For comparison, we show the same statistics for a variety of other expression experiments, which use different techniques (gene chips and SAGE). The figure shows that our results are largely consistent over the variety of experiments we analyzed. 
(By this, we mean the overall trend of high expression in the cytoplasm and low expression in the nucleus can be consistently observed in the different datasets. However, the datasets do differ in the exact value of each statistic, with these differences probably resulting from the slightly different protocols and growth conditions employed in each experiment.)

EXPRESSION FLUCTUATIONS. Expression measurements over a time course also enable us to relate fluctuations in expression to localization. As described in the caption to figure 1 and shown in the body of the figure, we measure the magnitude of fluctuation in terms of the standard deviation in expression ratio over a time course. For the yeast cell cycle timecourse3, 6 we find that secreted proteins have, perhaps predictably, high fluctuations. (However, proteins in the secretory pathway with final destinations in the ER or golgi show fluctuations slightly below average.)  Plasma membrane proteins, needed for transporting molecules out of the cell, have the second highest fluctuations. These results are biologically quite reasonable as the export of proteins out of the cell is quite variable and depends on the exact state of the cell, whereas the amount of intracellular proteins has to be maintained at a more constant level. 

For the diauxic shift timecourse2, which is also shown in figure 1, we again observe high fluctuations for secreted proteins. We also observe particularly high fluctuations for cytoplasmic and mitochondrial proteins, as expected when the cell shifts from fermentation to respiration and alters the activity of many metabolic enzymes. 

STRUCTURE-FUNCTION ANALYSIS. We can further understand the relationship between expression and localization by analyzing the functions of the associated proteins.  Using the MIPS classification9, we can subdivide the yeast genome into various functional categories – e.g. “cell structure” and “transcription.” Figure 3a shows the average absolute expression levels for each functional category for a variety of experiments, doing for function what figure 1 does for localization. Likewise, figure 3b shows box-plot representations of the expression distributions for select functional categories in a similar fashion to figure 2a.

We observe that proteins in the “transcription” and “transport” categories have lower average expression levels (1.8 and 2.7 versus 3.2 for all classified genes; all values are in copies per cell for the data set of Holstege et al.4; see figure 3a).  On the other hand, expression levels in the categories “protein synthesis” and “energy” are above average (16.0 and 5.4).  This is all in accord with the localization results (as noted above, nuclear and membrane proteins have low expression levels).  Proteins involved in transcription include DNA-binding and regulatory proteins with clear nuclear localization, and proteins involved in extracellular transport are often located in the cell membrane.  Furthermore, the high level of expression associated with protein synthesis is largely due to the ribosomal proteins (average expression level of 23.2), which are in the cytoplasm. (Notice, in contrast, that the amino-acyl-tRNA-synthetases, which are also part of the broad category of protein synthesis, have considerably lower level of expression.) Proteins involved in energy production include the cytoplasmic proteins involved in glycolysis, which have high levels of expression (20.5), and the lowly expressed mitochondrial proteins involved in the TCA cycle (2.0).  

The relationship between expression and localization is also linked to protein structure, although to a lesser extent.  Using membrane protein prediction plus classifications of the known soluble protein folds11, 12, we can subdivide the proteins in the yeast genome into helical membrane proteins, soluble proteins and, among the latter, proteins with all-a, all-b, and mixed ab structural architecture.  Figure 3c shows average expression levels for these structural classes, in analogy to the presentation in figures 1 and 3a for localization and function. We find a low average expression level for the transmembrane proteins (1.7).  On the other hand, proteins with mixed ab architecture, which are typically found in the cytosol13, are the most highly expressed among the soluble proteins (3.5 versus 2.5 for other soluble proteins).

Thus, to conclude, we find a clear statistical relationship between a gene’s expression level and its subcellular localization, with cytoplasmic proteins tending to be highly expressed and nuclear or membrane proteins more lowly expressed. This relationship may be useful in predicting subcellular localization, given expression information. The correlation between expression and localization may be related to the volumes of the various subcellular compartments.  The cytoplasm, for instance, has much more space for proteins than the other compartments.  In order to achieve the same effective concentration, the expression level for freely diffusing proteins destined for larger compartments may need to be higher than for smaller ones. In other words, genes associated with cytoplasmic proteins may be regulated differently, in a different dynamic range, than those associated with membrane and mitochondrial proteins. 

Acknowledgements
We thank the NIH and the Keck foundation for support.

References

1	Velculescu, V. E. et al. (1997) Characterization of the yeast transcriptome Cell 88, 243-51
2	DeRisi, J. L., Iyer, V. R. & Brown, P. O. (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale Science 278, 680-6
3	Cho, R. J. et al. (1998) A genome-wide transcriptional analysis of the mitotic cell cycle Mol Cell 2, 65-73
4	Holstege, F. C. et al. (1998) Dissecting the regulatory circuitry of a eukaryotic genome Cell 95, 717-28
5	Roth, F. P., Hughes, J. D., Estep, P. W. & Church, G. M. (1998) Finding DNA regulatory motifs within unaligned noncoding sequences clustered by whole-genome mRNA quantitation Nat Biotechnol 16, 939-45
6	Spellman, P. T. et al. (1998) Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization Mol Biol Cell 9, 3273-97
7	Jelinsky, S. A. & Samson, L. D. (1999) Global response of Saccharomyces cerevisiae to an alkylating agent Proc Natl Acad Sci U S A 96, 1486-91
8	Niehrs, C. & Pollet, N. Synexpression groups in eukaryotes Nature 402, 483-487 (1999)
9	Mewes, H. W. et al. (1999) MIPS: a database for genomes and protein sequences Nucleic Acids Res 27, 44-8
10	Hodges, P. E., McKee, A. H., Davis, B. P., Payne, W. E. & Garrels, J. I. (1999) The Yeast Proteome Database (YPD): a model for the organization and presentation of genome-wide functional data Nucleic Acids Res 27, 69-73
11	M Gerstein. (1998) Patterns of Protein-Fold Usage in Eight Microbial Genomes: A Comprehensive Structural Census Proteins 33: 518-534
12.	Jansen, R. & Gerstein M. (2000) Analysis of the Yeast Transcriptome with Broad Structural and Functional Categories: Characterizing Highly Expressed Proteins Nucleic Acids Res 28, 1481-1488
13	Hegyi, H. & Gerstein, M. (1999) The relationship between protein structure and function: a comprehensive survey with application to the yeast genome J Mol Biol 288, 147-64
14	Altschul, S. F. et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 25, 3389-402

Figure 1

Expression patterns of major compartments of the yeast cell.  The graph for each compartment consists of three parts (left, middle and right) described below.  All scales are logarithmic.

Left (solid) - Average absolute expression level of all transcripts in the compartment. Values refer to the data by Holstege et al.4, which we picked as a reference. 
The figure shows, for instance, that the average expression level is 14.4 copies/cell for transcripts in the cytoplasm, 2.4 for transcripts associated with proteins localized to the membrane, and so on.  We chose the Holstege data because we believe that it is the most accurate absolute gene expression level data. However, one would get similar results picking other datasets as a reference. In fact, on the website we show how similar results can be derived from a “combined” dataset derived from averaging together many different experiments. Note that further detail on this figure is shown in Figure 2, which gives the numbers of different transcripts for each compartment.

Middle - Fluctuation of expression levels during the timecourse of the yeast cell cycle with alpha-factor arrest6.  To calculate the fluctuation, we start with the logarithm of the expression ratio for gene i at time t: 

where r(i,t) and g(i,t) represent the red and green fluorescent signals at a particular timepoint.  Usually, the logarithm of the expression ratio is analyzed6 rather than the expression ratio because in general it is symmetrically distributed around zero.  Note that because of the structure of microarray experiments, all gene-to-gene comparisons have to be done using expression ratios rather than absolute measurements.  Then we calculate the standard deviation s(i)  in this quantity over all time points t for each gene i:

Finally, to get the numbers in the graph, we average s(i) over the genes in a given compartment.  

The results from the other cell cycle experiments with CDC156 and CDC283 show similar trends (data not shown).

Right – Fluctuation of expression levels during the timecourse of the diauxic shift experiment2.  Values are calculated in the same way as above.

Figure 2

Figure 2a: Figure 1 only shows the mean expression levels. To get some sense of the distribution of levels, we show a standard box plot representation of the distribution for the data set by Holstege et al4.  The box plot is explained in the inset.  The thick line in the middle of the central vertical box shows the median. The central box contains the values for 50% of the total number of transcripts around the mean. Thus, 25% transcripts have values above the upper boundary of the central box -- i.e. it is the 75% or top quartile line. Similarly, 25% transcripts have values below the lower boundary of the central box -- i.e. it is the 25% or bottom quartile line. The brackets extend up to 1.5 times the inter-quartile distance (the distance between the 25% and 75% lines) above and below the central box. Points beyond the brackets are defined as outliers. All measurements are in copies per cell. 

Figure 2b: In this subfigure we show key statistics derived from the box plot representation in part a. For comparative purposes we show these statistics for a variety of different experiments besides that of Holstege et al4.  The row “# ORFs” contains the number of different transcripts with expression levels in each experiment that are associated with the cellular compartment by cross-referencing with the localization databases. The row “75%” contains the expression value of the top quartile line, i.e. the 75% line. For instance, 75% transcripts belonging to the cytoplasm in the Holstege data have a value less than 24 copies per cell. Similarly, the row “50%” contains the median value of expression for transcripts in each compartment, and the row “25%” contains the expression value of the bottom quartile line. Although the experiments vary in methodology and growth conditions, the same trend can be observed. The differentiation of expression according to subcellular compartmentalization is evident in all cases.  More detailed statistics can be found on the website. All the data has been scaled into a common reference system based on the Holstege data. Furthermore, on the website, we give results similar to those in this figure for a “combined” dataset derived from averaging together many different experiments.

Figure 3
 
This figure shows statistics on gene expression related to functional and structural categories in analogy to the presentation relating expression to localization in figures 1 and 2. 

Figure 3a: Average expression levels of genes in selected MIPS9 categories.  The tree below the x-axis indicates a subset of the hierarchy of the different classifications -- e.g. it shows how all yeast proteins have as a subset “energy production” proteins, and how these, in turn, have subsets associated with glycolysis and the TCA cycle.  The y-axis displays the average expression levels for transcripts associated with each respective category.  We analyzed nine data sets1, 4, 5, 7, each represented by different symbols.  The columns represent the averages of all data sets.  The total number of ORFs in the dataset by Holstege et al.4 classified by MIPS is 2471 (“all classified”).  The number of transcripts for the other categories is 712 (transport), 721 (transcription), 318 (protein synthesis), 190 (ribosomal proteins), 37 (tRNA-synthetases), 210 (energy), 25 (glycolysis), and 19 (TCA cycle).

Figure 3b: Box plots of the expression level distributions for selected MIPS categories.  The box plots are again generated for the data set by Holstege et al4 and show the same distribution parameters as in figure 2a.

Figure 3c: Average expression levels of proteins in broad structural classes.  For structural classification we predicted, as described previously11, transmembrane segments in the yeast genome using a hydropathy scale and used PSI-blast14 to find matches of the yeast genome with PDB structures.  For the dataset by Holstege et al.4 the number of transcripts in each category are 600 (transmembrane), 4743 (soluble), 290 (all A), 202 (all B), and 781 (mixed A and B).  The number of transmembrane and soluble ORFs does not add up to the complete number of ORFs in the yeast genome because we are only considering ORFs that can be assigned to either category with high confidence.  The number of transcripts for the other structural classes reflects the amount of structure matches that could be found with the PDB.  More detailed statistics can be found at http://bioinfo.mbb.yale.edu/genome/expression.