Large- Scale Protein Localization in Yeast
Proteomics has contributed significantly to our knowledge of eukaryotic cell biology through largescale studies describing protein-protein interactions, protein complex composition, and relative protein abundance. Recent work in the budding yeast Saccharomyces cerevisiae (Kumar et al., 2002) has extended the scope of proteomics to encompass the genome-wide analysis of protein localizationmthe large-scale and systematic identification of the subcellular compartment or organelle to which a protein localizes. The subcellular localization of a protein can be of fundamental importance in characterizing the function and regulation of that protein. For example, an uncharacterized protein that localizes to the mitochondria may be inferred to function directly or indirectly in cellular respiration. Many transcription factors are regulated through subcellular compartmentalization: nuclear export of the transcription factor Pho4p is tightly regulated such that it is only localized within the nucleus (and therefore capable of activating transcription) in response to phosphate starvation (O'Neill et al., 1996). Protein function can also be addressed on a global scale by integrating protein localization data with a variety of other proteomic data sets. In particular, protein localization data are a strong complement to large-scale protein-protein interaction studies, corroborating many putative interactions while also highlighting potential false-positive results (Gerstein et al., 2002). Ultimately, genome-wide protein localization data will be helpful in identifying the constituent proteins of each cellular organelle and, potentially, mechanisms by which some of these proteins are regulated.
Yeast proteins may be localized through any of several approaches. By one common strategy, antibodies against a target protein can be used to immunolocalize that protein within a fixed cell. Antibodies against native proteins may be prepared by standard immunology; however, the feasibility of applying this approach on a proteome-wide scale has yet to be demonstrated. Alternatively, yeast proteins may be epitope tagged for subsequent immunolocalization. Tagged proteins can be localized by indirect immunofluorescence using monoclonal antibodies directed against the epitope tag. Epitope tags may be introduced into a protein-coding sequence by several methods. Many yeast vectors are available by which epitope tags may be fused to the amino (N) terminus or carboxy (C) terminus of a cloned gene (Longtine et al., 1998). In addition, epitope tags may be introduced at random within a protein-coding sequence by insertional mutagenesis using a modified epitope-bearing transposon (Hoekstra et al., 1991; Ross-Macdonald et al., 1997, 1999). A combination of random and directed tagging methods has been used successfully in Saccharomyces to epitope tag approximately 3600 yeast genes; subsequent immunolocalization of this protein set has defined the subcellular localization of nearly 2800 yeast proteins (Kumar et al., 2002).
As an alternative to immunolocalization, proteins may be localized by fluorescence microscopy using yeast open reading frame (ORF)-fluorescent protein chimeras. Coding sequence for a fluorescent protein [e.g., green fluorescent protein (GFP)] can be fused to a protein of interest using any number of appropriate tagging vectors (e.g., GFP-tagging vectors of the pFA6a series) (Longtine et al., 1998). Yeast ORF-GFP fusions may be constructed as integrated chromosomal alleles using these vectors in conjunction with standard polymerase chain reaction (PCR)-based methods. GFP tagging has been used extensively in S. cerevisiae for the localization of individual target proteins; ongoing efforts to tag all annotated yeast ORFs with GFP should enable large-scale analysis of protein localization by fluorescence microscopy in living yeast cells.
This article presents detailed protocols by which yeast ORFs may be epitope tagged and yeast strains containing tagged proteins prepared for immunofluorescence analysis in a 96-well format. The protocols presented here utilize the PCR-based tagging method of Longtine et al. (1997) to generate C-terminal chromosomal gene fusions with a sequence encoding three copies of the hemagglutinin (HA) epitope. The resulting HA-tagged yeast proteins can be immunolocalized by indirect immunofluorescence as described; the immunofluorescence protocol is adapted for performance in a 96-well format, facilitating proteome-scale analysis of protein localization.
II. MATERIALS AND INSTRUMENTATION
G418 sulfate (geneticin) may be purchased from Invitrogen Corp. (Cat. No. 11811023). Glusulase is a trademarked preparation of β-glucuronidase / sulfatase, each available from Sigma (Cat. Nos. G0258 and S8504). Zymolyase-100T is from Seikagaku America; Lyticase from Sigma may be substituted (Cat. No. L4025). The mouse anti-HA monoclonal antibody 16B12 is from Covance (Cat. No. MMS-101R-1000). Cy3-conjugated affinity-purified goat antimouse IgG is from the Jackson Laboratories (Cat. No. 115-165-146). 4,6-Diamidino-2-phenylindole (DAPI) is available from Sigma (Catalog No. D-9542). Poly-L-lysine is from Sigma (Cat. No. P1524). Microscope slides are from Carlson Scientific (Cat. No. 101805).
A. Epitope-Tagging Yeast Genes
Yeast genes may be epitope tagged through a variety of methods: the protocol described here is essentially that of Longtine et al. (1997) in which PCR is used to amplify a cassette from the vector pFA6a- 3HA-kanMX6 suitable for generating an integrated Cterminal HA-tagged allele of any nonrepeated yeast gene. The HA-tagging cassette (Fig. 1A) consists of a sequence encoding three copies of the HA epitope, the S. cerevisiae ADH1 terminator, and the kanMX6 module encoding resistance to G418. Tagging cassettes are amplified using primers with 5' ends corresponding to the target gene and 3' ends corresponding to the cassette; primer design is discussed later. Amplified cassettes are subsequently introduced into yeast by standard methods of DNA transformation (Gietz et al., 1992). By homologous recombination, the tagging cassette integrates at its intended genomic locus; transformants containing this cassette are selected on medium supplemented with G418. By this PCR-based approach, yeast genes may be systematically epitope tagged for subsequent large-scale immunolocalization.
Strains containing HA-tagged proteins may be immunolocalized by indirect immunofluorescence using monoclonal antibodies directed against the HA epitope (primary antibody) and the Cy3-conjugated secondary antibody. Immunolocalization in yeast necessitates permeabilization of the cell wall; here, yeast cells are spheroplasted using the trademarked enzyme preparations glusulase and Zymolyase-100T. The provided immunolocalization protocol is tailored for implementation in a 96-well format. Using this procedure, a single researcher can easily prepare 192 samples (two 96-well microtiter plates) for immunofluorescence in a period of 3 days (Kumar et al., 2000). Sample immunofluorescence patterns in cells containing HA-tagged proteins are presented in Fig. 2.
In designing protein localization studies, consideration should be paid to the placement of reporter/epitope tags relative to each target open reading frame. Typically, reporters and epitope tags are fused to either N or C termini of target genes; however, each tagging strategy possesses associated advantages and drawbacks. Organelle-specific targeting signals (e.g., mitochondrial targeting peptides and nuclear localization signals) are often located at the N terminus (Silver, 1991). N-terminal reporter fusions may disrupt these sequences, resulting in aberrant protein localizations. In other cases, C-terminal sequences may be important for proper function and regulation. For example, isoprenylation motifs are typically C-terminal, and sequences important for plasma membrane localization may be found throughout the gene-coding sequence (Nakai and Horton, 1999). While no single tagging strategy can be all-encompassing, a greater proportion of the yeast proteome should remain functional as C-terminal fusions: several studies have generated genome-wide Cterminal fusions for large-scale protein localization (Ding et al., 2000; Kumar et al., 2002).
Gene copy number may also impact upon the accuracy with which a tagged protein is localized. Overexpressed protein products can saturate intracellular transport mechanisms, potentially resulting in anomalous protein localization. However, weakly expressed single copy genes may yield insufficient protein levels for analysis, although the protocol presented here-in which tagged proteins are expressed endogenously under control of their native promoters-has been used successfully to localize nonabundant proteins (Kumar et al., 2002).
The immunofluorescence protocol presented here is reliable, but care should be taken to empirically determine optimal incubation times for spheroplasting (Zymolyase/glusulase treatment). The efficiency of spheroplasting may be monitored by phase-contrast microscopy. Ideally, cells should be a dark, translucent gray. Bright cells may have been insufficiently digested, whereas pale gray cells with little internal structure have likely been overdigested (Adams et al., 1998). In our hands, cells spheroplasted for 13-20min yield good staining patterns upon subsequent antibody treatment.
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