The protein architecture of the endocytic coat analyzed by FRET

Abstract

Endocytosis is a fundamental cellular trafficking pathway, which requires an organized assembly of the multiprotein endocytic coat to pull the plasma membrane into the cell. Although the protein composition of the endocytic coat is known, its functional architecture is not well understood. Here we determine the nanoscale organization of the endocytic coat by FRET microscopy in yeast. We assessed proximities of 18 conserved coat-associated proteins and used clathrin subunits and protein truncations as molecular rulers to obtain a high-resolution protein map of the coat. Furthermore, we followed rearrangements of coat proteins during membrane invagination and their binding dynamics at the endocytic site. We show that the endocytic coat is stratified into several functional layers situated above and below the clathrin lattice with key proteins transversing through the lattice deeply into the cytoplasm. We propose that this conserved design enables an efficient and regulated function of the endocytic coat during endocytosis.

FRET Microscopy in Yeast

“…The molecular architecture of several large multiprotein assemblies, which consist of dozens of subunits (usually presented in multiple copies), has been systematically analyzed by FRET in yeast: the organization of the nuclear pore complex and its associated transport factors [11,12], kinetochore [13][14][15], spindle pole body (microtubule organizing center in yeast) [16][17][18][19][20], cell division contractile ring [21] ( Figure 2B), and endocytic coat [22,23] ( Figure 2C). Also, interactions of several smaller complexes of DNA/chromatin regulatory proteins were determined by FRET: the architecture of yeast cohesin [24], interaction between Gal4 transcription factor and SAGA complex [25], crosstalk between PCNA protein Pol30 and SAS-I complex [26], or binding of ATR kinase complex Dcp2-Mec1 with PP4 phosphatase Psy2-Php3 [27].…”

“…Nuclear pore complex (NPC) sensitized emission CFP-YFP [11,12,42] Spindle pole body (SPB) sensitized emission acceptor photobleaching CFP-YFP mTq2-YFP [16][17][18][19][20] Kinetochore sensitized emission FLIM GFP-mCherry mTq2-YFP [13][14][15] Contractile ring acceptor photobleaching GFP/mNG-mCherry [21] Endocytic coat acceptor photobleaching GFP-mCherry mTq-mNG mNG-mScarlet [22] Cohesin sensitized emission CFP-YFP [24] SAGA-Gal4 transcription factor acceptor photobleaching spectral FRET CFP-YFP [25,43] PCNA-SAS-I complex FLIM CFP-YFP [26] ATR complex Dcp2-Mec1-PP4 phosphatase Psy2-Php3 sensitized emission GFP-RFP [27] Ste2 oligomerization spectral FRET CFP/GFP-YFP [28][29][30]44] Fet3-Ftr1 iron permease spectral FRET CFP-YFP [31] Ctr1 transporter oligomerization and copper binding spectral FRET CFP-YFP [32] Ato1-Ato2 proteins acceptor photobleaching, FLIM GFP-tdimer2 CFP-Venus [33] V-ATPase disassembly sensitized emission CFP-YFP [34] Tom70 oligomerization sensitized emission CFP-YFP [35] CDK inhibitor Sic1-cyclins FLIM mCerulean-YFP [36] Ste5-Fus3 interaction acceptor photobleaching GFP-mStrawberry [37] Prp prion aggregation donor photobleaching CFP-YFP [38] Prp-amyloid β interaction acceptor photobleaching CFP-YFP [39] HTT huntingtin aggregation acceptor photobleaching CFP-Venus [40] Toh1 aggregation with Rnq1 and Sup35 prion proteins acceptor photobleaching CFP-YFP [41] 1 For individual FRET techniques and fluorescent proteins, see please the main text. 2 FRET-based protein proximity mapping in yeast can also encounter some difficulties.…”

“…A critical drawback of this method is its endpoint character caused by irreversible photodestruction of the acceptor fluorophore. Nevertheless, FRET changes in time (e.g., expected change of protein complex composition or conformation) can be followed indirectly, for example by using another protein as a "time stamp" of the process (see [19,22] for examples). To capture transient molecular proximities, cells can first be fixed with formaldehyde, which was recently shown to preserve temporal protein complexes, as well as FRET between FPs [22,85].…”

“…The blue shift of its excitation spectrum also increases its spectral overlap with CFPs. According to comprehensive tests in other systems [93] and in our practice [22,66] in yeast, mTq2-mNG constitutes a very robust FRET pair for many FRET techniques.…”