Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore.
Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.
Ohba
Tomoyuki
T
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Schirmer
Eric C
EC
Nishimoto
Takeharu
T
Gerace
Larry
L
D000255:Adenosine Triphosphate / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000502:physiology; D004734:Energy Metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology*; D034881:Nuclear Lamina / Q000187:drug effects / Q000502:physiology; D022022:Nuclear Pore / Q000502:physiology*; D028861:Nuclear Pore Complex Proteins / Q000502:physiology; D009687:Nuclear Proteins / Q000502:physiology*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D020123:Sirolimus / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D000255:Adenosine Triphosphate; D020123:Sirolimus