Unifying Fluorescence Microscopy and Mass Spectrometry for Studying Protein Complexes in Cells

Abstract

We have developed and applied a method unifying fluorescence microscopy and mass spectrometry for studying spatial and temporal properties of proteins and protein complexes in yeast cells. To combine the techniques, first we produced a variety of DNA constructs that can be used for genomic tagging of proteins with modular fluorescent and affinity tags. The modular tag consists of one of the multiple versions of monomeric fluorescent proteins fused to a variety of small affinity epitopes. After this step we tested the constructs by tagging two yeast proteins, Pil1 and Lsp1, the core components of eisosomes, the large protein complexes involved in endocytosis in Saccharomyces cerevisiae, with a variety of fluorescent and affinity probes. Among the modular tags produced we found several combinations that were optimal for determining subcellular localization and for purifying the tagged proteins and protein complexes for the detailed analysis by mass spectrometry. And finally, we applied the designed method for finding the new protein components of eisosomes and for gaining new insights into molecular mechanisms regulating eisosome assembly and disassembly by reversible phosphorylation and dephosphorylation. Our results indicate that this approach combining fluorescence microscopy and mass spectrometry into a single method provides a unique perspective into molecular mechanisms regulating composition and dynamic properties of the protein complexes in living cells.

Formation and stability of eisosomes in the filamentous fungus Ashbya gossypii

“…Several functionally partially redundant components, such as Slm1 and Slm2 (Fadri et al, 2005), Pkh1 and Pkh2 (Casamayor et al, 1999;Luo et al, 2008;Walther et al, 2007), and Nce102 and Fhn1 (Loibl et al, 2010), are encoded by only one gene in A. gossypii. Likewise only a single syntenic gene is present in the A. gossypii genome for the fairly uncharacterized twin genes YMR031c and YKL050c, and YMR086w and YKL105c (Deng et al, 2009;Frohlich et al, 2009). For most A. gossypii genes listed in Table 1, we found moderate to high mRNA levels in spores.…”

“…In the absence of ScPil1, eisosome biogenesis is disrupted and the remaining According to three publications (Deng et al, 2009;Frohlich et al, 2009;Grossmann et al, 2008). Two gene names are listed for twins (duplicated genes still present from the whole genome duplication in the S. cerevisiae precursor).…”

Furrow-like invaginations of the yeast plasma membrane correspond to membrane compartment of Can1

“…Based on confocal localizations of Can1-GFP and Pma1-GFP, the size of the MCC patches was estimated to be ~300 nm. To date, more than 20 cortical proteins have been found to colocalize with the MCC, nine of which are integral to the plasma membrane (Grossmann et al, 2008;Maier et al, 2008;Deng et al, 2009). In addition to Can1, three other permeases with targeting pathways that are dependent on specific lipids, Fur4, Tat2 and heterologous HUP1 (Chlorella kessleri), were identified as MCC constituents (Malinska et al, 2004;Grossmann et al, 2006;Grossmann et al, 2007).…”

Reassessment of the role of plasma membrane domains in the regulation of vesicular traffic in yeast

“…For simplicity, we will use here the term MCC to collectively refer to the membrane furrows and all associated transmembrane and cytosolic proteins. In total 22 proteins (9 transmembrane proteins and 13 cytosolic proteins) have been shown to localize to this domain (Deng et al, 2009;Grossmann et al, 2008). A key protein in the maintenance of MCC organization is Pil1.…”