Analysis of membrane proteins localizing to the inner nuclear envelope in living cells.
Understanding the protein composition of the inner nuclear membrane (INM) is fundamental to elucidating its role in normal nuclear function and in disease; however, few tools exist to examine the INM in living cells, and the INM-specific proteome remains poorly characterized. Here, we adapted split green fluorescent protein (split-GFP) to systematically localize known and predicted integral membrane proteins in Saccharomyces cerevisiae to the INM as opposed to the outer nuclear membrane. Our data suggest that components of the endoplasmic reticulum (ER) as well as other organelles are able to access the INM, particularly if they contain a small extraluminal domain. By pairing split-GFP with fluorescence correlation spectroscopy, we compared the composition of complexes at the INM and ER, finding that at least one is unique: Sbh2, but not Sbh1, has access to the INM. Collectively, our work provides a comprehensive analysis of transmembrane protein localization to the INM and paves the way for further research into INM composition and function.
Stowers Institute for Medical Research, Kansas City, MO 64110.
Christine J
http://orcid.org/0000-0002-6932-8560
CJ
Smoyer
Stowers Institute for Medical Research, Kansas City, MO 64110.
Santharam S
http://orcid.org/0000-0003-2579-8904
SS
Katta
Stowers Institute for Medical Research, Kansas City, MO 64110.
Jennifer M
JM
Gardner
Stowers Institute for Medical Research, Kansas City, MO 64110.
Lynn
L
Stoltz
Stowers Institute for Medical Research, Kansas City, MO 64110.
Scott
S
McCroskey
Stowers Institute for Medical Research, Kansas City, MO 64110.
William D
http://orcid.org/0000-0001-8302-8305
WD
Bradford
Stowers Institute for Medical Research, Kansas City, MO 64110.
Melainia
http://orcid.org/0000-0002-8947-9841
M
McClain
Stowers Institute for Medical Research, Kansas City, MO 64110.
Sarah E
SE
Smith
Stowers Institute for Medical Research, Kansas City, MO 64110.
Brian D
BD
Slaughter
Stowers Institute for Medical Research, Kansas City, MO 64110.
Jay R
JR
Unruh
Stowers Institute for Medical Research, Kansas City, MO 64110 slj@stowers.org.|Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160.
Sue L
http://orcid.org/0000-0001-8312-7063
SL
Jaspersen
D004721:Endoplasmic Reticulum / Q000378:metabolism; D016681:Genome, Fungal; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism*; D000072417:Protein Domains; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins