Novel myristoylation of the sperm-specific hexokinase 1 isoform regulates its atypical localization

Abstract

The hexokinase 1 variant in mammalian spermatozoa (HK1S) has a unique N-terminus and this isoform atypically localizes to the plasma membrane. However, the mechanism of this process currently remains ambiguous. In this report, we show that fatty acylation underlies the specific sorting of HK1S. Employing chimeric reporter constructs, we first established that compartmentalization of HK1S does not function exclusively in sperm cells and that this feature is swappable to somatic HEK293 cells. Although the N-terminus lacks the classical consensus signature for myristoylation and the sequence-based predictions fail to predict myristoylation of HK1S, complementary experimental approaches confirmed that HK1S is myristoylated. Using live-cell confocal microscopy, we show that the mutation of a single amino acid, the myristoyl recipient Gly2, impedes the prominent feature of plasma membrane association and relocates the enzyme to the cytosol but not the nucleus. Additionally, substitutions of the putatively palmitoylated Cys5 is also reflected in a similar loss of compartmentalization of the protein. Taken together, our findings conclusively demonstrate that the N-terminal ‘MGQICQ’ motif in the unique GCS domain of HK1S acquires hydrophobicity by dual lipidic modifications, N-myristoylation and palmitoylation, to serve the requirements for membranous associations and thus its compartmentalization.

Quantitative proteomic analysis of sperm in unexplained recurrent pregnancy loss

“…Cellular energy metabolism is correlated with cell fate and a few studies report the association between glycogen and the nuclear envelope, the endoplasmic reticulum, as well as the annulate lamellae of embryonic and transformed cells [29, 30]. HK1 protein localizes on the outer membrane of mitochondria and phosphorylates glucose to produce glucose-6-phosphate, which is the first step of glucose metabolism pathways [31]. Spermatogenic cell-specific type 1 hexokinase (HK1S), an isoform of HK1, is found abundantly in sperm, which is mainly expressed in area of the sperm flagellum [32].…”

A Not-So-Ancient Grease History: Click Chemistry and Protein Lipid Modifications

“…The NMT2-mediated N- myristoylation of mouse LMCD1, as evidenced by Az-C12 labeling, reversed its repressor activity, underscoring the differential roles of LMCD1 transactivation activity in humans versus mice . Furthermore, N -myristoylation regulates the cell type-specific sorting of proteins such as the spermatozoan variant Hexokinase 1 (HK1S) . Cell-free translation combined with cotranslational labeling with Az-C12 and subsequent Staudinger detection confirmed HK1S N -myristoylation, and the presence of myristate directs it to the plasma membrane, which is not the case in other cell types.…”

“…324 Furthermore, N-myristoylation regulates the cell type-specific sorting of proteins such as the spermatozoan variant Hexokinase 1 (HK1S). 325 Cell-free translation combined with cotranslational labeling with Az-C12 and subsequent Staudinger detection confirmed HK1S N-myristoylation, and the presence of myristate directs it to the plasma membrane, which is not the case in other cell types.…”