12th Carl Zeiss sponsored workshop on ... - Institut Fresnel

Different acylation motifs direct multiply orthogonoal co-localization of lipid anchored
proteins in live cell membranes
J.T. Groves*, M.B. Forstner*, B.F. Lillemeier**, and M.M. Davis**
* Howard Hughes Medical Institute
University of California Berkeley and Lawrence Berkeley National Laboratory
** Stanford University
Contact author: JTGroves@lbl.gov
A number of chemically distinct genetically-encoded acylation modifications link soluble
protein domains to cell membranes in vivo. It is widely speculated that these provide targeting
mechanisms that direct protein organization in the membrane. Here, we use fluorescence
cross correlation spectroscopy to probe interactions among all combinatorial pairs of four
common lipid anchoring motifs expressed in live primary T cells as fluorescent protein
fusions. All homogeneous anchor combinations lead to strong co-localization of the anchored
proteins into small mobile clusters on the membrane surface. In contrast, results from
heterogeneous anchor pairings reveal mutual avoidance among each of the three cytoplasmic
membrane leaflet anchors examined (palmityl:palmityl:myristoyl from Lck, myristoyl from
Src, and gerenyl-gerenyl from RhoA). The extracellular leaflet GPI anchor co-localizes with
the Lck anchor while avoiding the other anchors types. Both cholesterol depletion and actin
cytoskeleton disruption interfere with anchor-mediated clustering. These results indicate that
protein acylation can direct co-localization into at least three orthogonally composed small
dynamic clusters on the membrane surface. Additionally, at least one of these cluster types
exhibits trans-bilayer structure.
Fig. 1. A small combinatorial library of different genetically encoded lipid anchor sequences
fused to red or green fluorescent proteins is used to explore anchor co-localization in the
membranes of live primary T cells.
12 th <strong>Zeiss</strong> <strong>sponsored</strong> FCS <strong>workshop</strong> – Cargèse, Corsica, France – <strong>12th</strong> -16th October 2009