Stepwise ATP translocation into the endoplasmic reticulum by human SLC35B1

Area of Science:

• Cellular Biology
• Molecular Transport
• Biochemistry

Background:

• Mitochondria generate ATP, crucial for cellular functions.
• The endoplasmic reticulum (ER) requires imported ATP for protein homeostasis and trafficking.
• The ER-resident ATP transporter remained elusive, with SLC35B1 (AXER) recently proposed as a candidate.

Purpose of the Study:

• To investigate the function of human SLC35B1 (AXER) as an ER ATP importer.
• To elucidate the transport mechanism and structural basis of SLC35B1 activity.
• To validate the physiological relevance of SLC35B1 in cellular growth.

Main Methods:

• Biochemical assays to assess nucleotide binding and transport kinetics.
• CRISPR-Cas9 gene editing to create SLC35B1 knockout cell lines.
• Cryogenic electron microscopy (cryo-EM) to determine high-resolution structures of SLC35B1.

Main Results:

• Human SLC35B1 binds ATP/ADP, not nucleotide sugars, and facilitates ATP import into ER microsomes.
• SLC35B1 is essential for cell growth, confirmed by knockout studies.
• Seven cryo-EM structures reveal ATP/ADP translocation via a stepwise mechanism involving vertical nucleotide repositioning within a flexible binding site.

Conclusions:

• SLC35B1 functions as a dedicated ER ATP importer, crucial for cellular energy balance.
• The stepwise translocation mechanism of SLC35B1 represents a novel mode of substrate transport for SLC transporters.
• Understanding SLC35B1's function provides insights into ER energetics and cellular health.