Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Summary

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.

Abstract

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Introduction

Deregulation of lipid metabolism is a common characteristic of cancer and metabolic diseases^1-6. In these processes, sterol regulatory element-binding proteins (SREBPs), a family of transcription factors, play a critical role in controlling the expression of genes important for the uptake and synthesis of cholesterol, fatty acids, and phospholipids^7-10.

SREBPs, including SREBP-1a, SREBP-1c, and SREBP-2, are synthesized as inactive precursors that bind to the endoplasmic reticulum (ER) membrane by virtue of two transmembrane domains⁷. The N-terminus of SREBPs contains a DNA-binding domain for the transactivat....

Protocol

1. Detection of Endogenous SCAP Protein in Human Cells

1. Cell Culture and Treatment
   1. Seed ~ 1 × 10⁶ U87 cells in a 10 cm dish with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and incubate the cells at 37 °C and 5% CO₂ for 24 hr before treatment.
   2. Wash cells once with phosphate-buffered saline (PBS), then switch cells to fresh DMEM medium with or without glucose (5 mM) for 12 hr.
2. Preparation of Cell Membrane Fractions and Nuclear Extracts
   1. Wash cells once with PBS, scrape into 1 ml PBS, and centrifuge at 1,000 x g for 5 min at ....

Results

Figure 1 shows the detection of endogenous SCAP protein and SREBP-1 nuclear form in human glioblastoma U87 cells in response to glucose stimulation by using western blot. The membrane protein SCAP is detected in the "membrane fraction". The nucleus form (N-terminal) of SREBP-1 is detected in the "nuclear extracts". The level of SCAP protein (PDI serves as an internal control of ER membrane proteins) and SREBP-1 nuclear form are markedly enhanced by glucose stimulatio.......

Discussion

In this study, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using GFP-labeling and confocal microscopy. The method is specifically used to analyze membrane protein and is an important tool to investigate SCAP N-glycosylation and trafficking.

Compared with the method.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
X-treme GENE HP DNA Transfection Reagent	Roche	6366236001
Opti-MEMI medium	Life Technologies	31985-070
trypsin	Sigma	T6567
soybean trypsin inhibitor	Sigma	T9777
PNGase F	Sigma	P7367
Anti-SCAP (9D5) antibody	Santa Cruz	sc-13553
GFP antibody	Roche	11814460001
SREBP-1 antibody (IgG-2A4)	BD Pharmingen	557036
PDI Antibody (H-17)	Santa Cruz	sc-30932
Lamin A Antibody (H-102)	Santa Cruz	sc-20680
SCAP antibody (a.a 450-500)	Bethyl Laboratories, Inc.	A303-554A
Dulbecco’s modified Eagle’s medium (DMEM) without glucose, pyruvate and glutamine	Cellgro	17-207-CV	Add 1 mM Pyravate and 4 mM Glutamine before use
22 G x 1 1/2 needle	BD	305156
pepstatin A	Sigma	P5318
leupeptin	Sigma	L2884
PMSF	Sigma	P7626
DTT	Sigma	43819
ALLN	Sigma	A6185
Nitrocellulose membrane	GE	RPN3032D
EDTA Solution 0.5 M pH 8.5 100 ml	VWR	82023-102
EGTA 0.5 M sterile (pH 8.0) 50 ml	Fisher Scentific	50255956
HyClone FBS	Thermo scientific	SH3007103
glycerol	Sigma	G5516
β-mercaptoethanol	Sigma	M3148
bromophenol blue	Sigma	B8026
prolong gold antifade reagent with dapi	Life Technologies	P36935
L-glutamine (200 mM)	Life Technologies	25030081
sodium pyruvate	Life Technologies	11360-070