As UW-Madison researcher Xingmin Zhang finishes his lunch and heads back into lab, the cells in his pancreas are already hard at work sending out insulin to convert that food into the energy his body needs to finish up the day’s experiments.
Hormones, like insulin, are secreted from cells in a tightly regulated manner with the help of many proteins. Zhang, a graduate student from Dr. Tom Martin’s biochemistry lab, identified and studied a new protein involved in the processes that transport proteins to the plasma membrane for secretion. The results, published this month in The Journal of Cell Biology, highlight how the proper flow of traffic both to and from the plasma membrane is necessary for efficient secretion. Impaired insulin secretion ultimately leads to diabetes.
“It helps to have mechanistic insight into a cellular process to then begin to understand what is going wrong in a disease state,” said Zhang.
On their way to the plasma membrane, proteins destined for secretion make a few stops in different compartments within the cell. Like all other proteins, they are synthesized in the cytoplasm, but then are immediately move into the endoplasmic reticulum. Then they travel to the Golgi apparatus, which functions as the post office for cell, sorting proteins into small cellular transport boxes called vesicles destined for specific locations. Hormones are packaged into dense core vesicles that bud off the Golgi apparatus.
Key steps must occur to make these vesicles ready for secretion as they travel to the plasma membrane. First, the cargo contained within the vesicles needs to be refined as a quality control measure, ensuring that all those proteins are in the appropriate “box.” This cargo refinement occurs via a class of smaller cellular transport boxes called endosomes. Additionally, the inside of the vesicle becomes more acidic, making the proteins biologically active.
Once the vesicles reach the plasma membrane, a group of proteins that respond to calcium help the vesicle fuse to the plasma membrane, resulting in the dumping of the vesicle’s contents outside the cell. This subset of proteins then needs to be recycled in endosomes back to the Golgi apparatus for successive waves of vesicle fusion to occur.
Cell biologists have established that many of the proteins mediating vesicle fusion rely on the same region of the protein, so Zhang designed a method in a cell line to assess the ability of the cells to secrete in the absence of each protein containing this region. Several proteins identified in this manner already had known roles in protein trafficking and secretion, but many others did not.
Zhang focused his efforts on ascertaining the role of one of those proteins of unknown function, BAIAP3. BAIAP3 seemed to be a promising candidate for follow-up study because it was abundantly present in the regions of the brain which signal to other tissues via secretion of hormones and other signaling proteins. Zhang also discovered that BAIAP3 was expressed in the beta cells of the pancreas and that eliminating its expression in a beta cell line blunted insulin secretion, making him more confident in its role in protein trafficking.
Zhang knew that to figure out how BAIAP3 was involved in protein trafficking he had to know its address in the cell. But, naturally, this turned out to be the hardest aspect of the study.
Microscopes enable scientists to see where their protein is within a cell. Proteins known to reside in a specific compartment are landmarks of that location. Scientists color-code different proteins within the cell using markers to identify either the protein itself or a small region of the protein called a tag. Zhang encountered a problem many researchers face at some point: finding a marker that faithfully labeled BAIAP3. He tried adding a tag onto the protein since the markers that recognize tags are more accurate, only to find that the tag was interfering with the protein’s function.
After months of troubleshooting, Zhang finally found that BAIAP3 was in endosomes. He now needed another completely different method to be sure of this finding, but again, he ran into difficulties. He struggled with published protocols detailing the steps required to crack open cells and separate the different subcellular compartments, having to methodically optimize his own protocol before confirming that BAIAP3 is indeed localized on endosomes.
Zhang used this information to infer that BAIAP3 was involved in recycling proteins from the plasma membrane to the Golgi apparatus to support continuing levels of secretion. He then demonstrated that BAIAP3 interacts with proteins known to mediate the fusion of endosomes with the Golgi apparatus in a calcium dependent manner.
Zhang’s work emphasizes that protein trafficking back from the plasma membrane is critical to maintaining a steady flow of traffic towards it. This study adds to the growing body of literature describing the significance of this trafficking route, outlining another possible avenue through which hormone and neuropeptide secretion can go awry.
Having just defended his PhD thesis in April, Zhang is now busily interviewing for postdoctoral fellowship positions across the country, looking to do more translational research.
“Secretion mediates a lot of cellular processes, so I’m glad to have gotten a good training in cell biology,” said Zhang. “It’s a great foundation upon which to learn new skills.”