Membrane Topology of Human Presenilin-1 in SK-N-SH Cells Determined by Fluorescence Correlation Spectroscopy and Fluorescent Energy Transfer

Krishna Midde, Ryan Rich, Ashwini Saxena, Ignacy Gryczynski, Julian Borejdo, Hriday Das

Research output: Contribution to journalArticleResearchpeer-review

5 Citations (Scopus)

Abstract

Presenilin-1 (PS1) protein acts as passive ER Ca2+ leak channels that facilitate passive Ca2+ leak across ER membrane. Mutations in the gene encoding PS1 protein cause neurodegeneration in the brains of patients with familial Alzheimer’s disease (FAD). FADPS1 mutations abrogate the function of ER Ca2+ leak channel activity in human neuroblastoma SK-N-SH cells in vitro (Das et al., J Neurochem 122(3):487–500, 2012) and in mouse embryonic fibroblasts. Consequently, genetic deletion or mutations of the PS1 gene cause calcium (Ca2+) signaling abnormalities leading to neurodegeneration in FAD patients. By analogy with other known ion channels it has been proposed that the functional PS1 channels in ER may be multimers of several PS1 subunits. To test this hypothesis, we conjugated the human PS1 protein with an NH2-terminal YFP-tag and a COOH-terminal CFP-tag. As expected YFP–PS1, and PS1–CFP were found to be expressed on the plasma membranes by TIRF microscopy, and both these fusion proteins increased ER Ca2+ leak channel activity similar to PS1 (WT) in SK-N-SH cells, as determined by functional calcium imaging. PS1–CFP was either expressed alone or together with YFP–PS1 into SK-N-SH cell line and the interaction between YFP–PS1 and PS1–CFP was determined by Förster resonance energy transfer analysis. Our results suggest interaction between YFP–PS1 and PS1–CFP confirming the presence of a dimeric or multimeric form of PS1 in SK-N-SH cells. Lateral diffusion of PS1–CFP and YFP–PS1 in the plasma membrane of SK-N-SH cells was measured in the absence or in the presence of glycerol by fluorescence correlation spectroscopy to show that both COOH-terminal and NH2-terminal of human PS1 are located on the cytoplasmic side of the plasma membrane. Therefore, we conclude that both COOH-terminal and NH2-terminal of human PS1 may also be oriented on the cytosolic side of ER membrane.

Original languageEnglish
Pages (from-to)923-932
Number of pages10
JournalCell Biochemistry and Biophysics
Volume70
Issue number2
DOIs
StatePublished - 2 Oct 2014

Fingerprint

Presenilin-1
Fluorescence Spectrometry
Energy Transfer
Energy transfer
Fluorescence
Topology
Spectroscopy
Membranes
Cell membranes
Cell Membrane
Alzheimer Disease
Calcium
Mutation
Proteins
Gene encoding
Calcium Signaling
Sequence Deletion
Fibroblasts
human PSEN1 protein
Neuroblastoma

Keywords

  • FCS
  • FRET
  • Lateral diffusion
  • Membrane topology
  • Presenilin-1
  • TIRF microscopy

Cite this

@article{493edafe981e473ba68bff26e2748090,
title = "Membrane Topology of Human Presenilin-1 in SK-N-SH Cells Determined by Fluorescence Correlation Spectroscopy and Fluorescent Energy Transfer",
abstract = "Presenilin-1 (PS1) protein acts as passive ER Ca2+ leak channels that facilitate passive Ca2+ leak across ER membrane. Mutations in the gene encoding PS1 protein cause neurodegeneration in the brains of patients with familial Alzheimer’s disease (FAD). FADPS1 mutations abrogate the function of ER Ca2+ leak channel activity in human neuroblastoma SK-N-SH cells in vitro (Das et al., J Neurochem 122(3):487–500, 2012) and in mouse embryonic fibroblasts. Consequently, genetic deletion or mutations of the PS1 gene cause calcium (Ca2+) signaling abnormalities leading to neurodegeneration in FAD patients. By analogy with other known ion channels it has been proposed that the functional PS1 channels in ER may be multimers of several PS1 subunits. To test this hypothesis, we conjugated the human PS1 protein with an NH2-terminal YFP-tag and a COOH-terminal CFP-tag. As expected YFP–PS1, and PS1–CFP were found to be expressed on the plasma membranes by TIRF microscopy, and both these fusion proteins increased ER Ca2+ leak channel activity similar to PS1 (WT) in SK-N-SH cells, as determined by functional calcium imaging. PS1–CFP was either expressed alone or together with YFP–PS1 into SK-N-SH cell line and the interaction between YFP–PS1 and PS1–CFP was determined by F{\"o}rster resonance energy transfer analysis. Our results suggest interaction between YFP–PS1 and PS1–CFP confirming the presence of a dimeric or multimeric form of PS1 in SK-N-SH cells. Lateral diffusion of PS1–CFP and YFP–PS1 in the plasma membrane of SK-N-SH cells was measured in the absence or in the presence of glycerol by fluorescence correlation spectroscopy to show that both COOH-terminal and NH2-terminal of human PS1 are located on the cytoplasmic side of the plasma membrane. Therefore, we conclude that both COOH-terminal and NH2-terminal of human PS1 may also be oriented on the cytosolic side of ER membrane.",
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Membrane Topology of Human Presenilin-1 in SK-N-SH Cells Determined by Fluorescence Correlation Spectroscopy and Fluorescent Energy Transfer. / Midde, Krishna; Rich, Ryan; Saxena, Ashwini; Gryczynski, Ignacy; Borejdo, Julian; Das, Hriday.

In: Cell Biochemistry and Biophysics, Vol. 70, No. 2, 02.10.2014, p. 923-932.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Membrane Topology of Human Presenilin-1 in SK-N-SH Cells Determined by Fluorescence Correlation Spectroscopy and Fluorescent Energy Transfer

AU - Midde, Krishna

AU - Rich, Ryan

AU - Saxena, Ashwini

AU - Gryczynski, Ignacy

AU - Borejdo, Julian

AU - Das, Hriday

PY - 2014/10/2

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AB - Presenilin-1 (PS1) protein acts as passive ER Ca2+ leak channels that facilitate passive Ca2+ leak across ER membrane. Mutations in the gene encoding PS1 protein cause neurodegeneration in the brains of patients with familial Alzheimer’s disease (FAD). FADPS1 mutations abrogate the function of ER Ca2+ leak channel activity in human neuroblastoma SK-N-SH cells in vitro (Das et al., J Neurochem 122(3):487–500, 2012) and in mouse embryonic fibroblasts. Consequently, genetic deletion or mutations of the PS1 gene cause calcium (Ca2+) signaling abnormalities leading to neurodegeneration in FAD patients. By analogy with other known ion channels it has been proposed that the functional PS1 channels in ER may be multimers of several PS1 subunits. To test this hypothesis, we conjugated the human PS1 protein with an NH2-terminal YFP-tag and a COOH-terminal CFP-tag. As expected YFP–PS1, and PS1–CFP were found to be expressed on the plasma membranes by TIRF microscopy, and both these fusion proteins increased ER Ca2+ leak channel activity similar to PS1 (WT) in SK-N-SH cells, as determined by functional calcium imaging. PS1–CFP was either expressed alone or together with YFP–PS1 into SK-N-SH cell line and the interaction between YFP–PS1 and PS1–CFP was determined by Förster resonance energy transfer analysis. Our results suggest interaction between YFP–PS1 and PS1–CFP confirming the presence of a dimeric or multimeric form of PS1 in SK-N-SH cells. Lateral diffusion of PS1–CFP and YFP–PS1 in the plasma membrane of SK-N-SH cells was measured in the absence or in the presence of glycerol by fluorescence correlation spectroscopy to show that both COOH-terminal and NH2-terminal of human PS1 are located on the cytoplasmic side of the plasma membrane. Therefore, we conclude that both COOH-terminal and NH2-terminal of human PS1 may also be oriented on the cytosolic side of ER membrane.

KW - FCS

KW - FRET

KW - Lateral diffusion

KW - Membrane topology

KW - Presenilin-1

KW - TIRF microscopy

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DO - 10.1007/s12013-014-9999-z

M3 - Article

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JO - Cell Biochemistry and Biophysics

JF - Cell Biochemistry and Biophysics

SN - 1085-9195

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