Figure 5.

A carboxymethyl-dependent lamin receptor in the nuclear envelope. (A) NLS-YFP-C13 (green in merge), representing the terminal 13 residues of lamin B1, was expressed in HeLa cells; the cells were fixed and labeled with 8D1 (red in merge). A single confocal section is shown. NLS-YFP-C13 localized throughout the endomembrane system and plasma membrane. The absence of 8D1 reactivity confirms that the construct was not proteolyzed despite membrane association. (B) NLS-YFP-C40 (green in merge) expressed in HeLa cells localized to the nuclear envelope and was 8D1 positive (red in merge), confirming that CAAX processing took place. A single confocal section is shown. (C) Single confocal sections through the mid-nucleus of wild-type and knockout cell lines, deficient in individual CAAX-processing enzymes, expressing NLS-YFP-C40, show failure to localize to the nuclear envelope in Rce1−/− and Icmt−/− cells. The nuclear envelope localization of NLS-YFP-C40 was not affected in Zmpste24−/− cells. Intranuclear levels of the expressed protein were more pronounced in Icmt−/−cells compared with Rce1−/− cells. (D) Farnesylation and membrane association was not impaired in Rce1- or Icmt-deficient cells. NLS-YFP-C40 is partially colocalized (merge) with rhodamine–concanavalin A in Icmt−/− cells (not depicted) and Rce1−/− cells. Bars, 10 μm.